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The Journal of Immunology, 2002, 169: 3307-3313.
Copyright © 2002 by The American Association of Immunologists

HIV-1 Transcription and Virus Production Are Both Accentuated by the Proinflammatory Myeloid-Related Proteins in Human CD4+ T Lymphocytes1

Carle Ryckman2, Gilles A. Robichaud2, Jocelyn Roy2, Réjean Cantin, Michel J. Tremblay3 and Philippe A. Tessier3

Centre de Recherche en Infectiologie, Pavillon Centre Hospitalier de l’Université Laval, Centre Hospitalier Universitaire de Québec, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
S100A8, S100A9, and S100A12, collectively known as myeloid-related proteins (MRPs), are highly expressed by the myeloid cell lineage and are found in the extracellular milieu during infections and inflammatory conditions. Recent data showed high levels of MRPs in the serum of HIV type 1 (HIV-1)-infected patients which correlated with disease progression and low CD4+ counts. Therefore, we set out to investigate the effect of MRPs on HIV-1 replication. We observed a 4- to 5-fold induction of virus production in J1.1, a human T lymphoid cell line latently infected with HIV-1, following treatment with MRPs. Using luciferase-based reporter gene assays, we demonstrated that MRPs induce a dose- and time-dependent activation of the HIV-1 long terminal repeat promoter region that could be blocked by specific anti-MRP polyclonal Abs and by physical denaturation of these proteins. The MRP-mediated induction was acting through the HIV-1 enhancer sequence and was dependent upon NF-{kappa}B activity. These latter results were also confirmed by EMSA experiments conducted in Jurkat cells and freshly isolated PBMCs. In conclusion, we demonstrate that MRPs induce HIV-1 transcriptional activity and viral replication in infected CD4+ T-lymphocytes at concentrations similar to those found in the serum of HIV-1-infected patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Opportunistic infections and inflammatory situations frequently result in an enhanced viral load in individuals infected with HIV type 1 (HIV-1).4 Various cytokines and soluble factors are released in the serum of these patients during opportunistic pathologies, some of which may directly contribute to the virus replicative cycle by positively affecting the regulatory elements of HIV-1 (i.e., the long terminal repeat (LTR)) (1, 2, 3). Virus production is thus influenced by the microenvironment that is surrounding virus-infected cells.

The myeloid-related proteins (MRPs) S100A8, S100A9, and S100A12 are calcium-binding proteins that belong to the S100 protein family (4, 5). Expression of the MRPs is mostly confined to neutrophils, monocytes (5, 6), and activated macrophages (7, 8, 9, 10). They are also expressed by certain epithelial cells (11, 12), activated endothelial cells (13), and keratinocytes (14, 15). S100A8 and S100A9 associate noncovalently to form homodimers and the heterodimer S100A8/A9 (16, 17, 18). MRPs have been shown to exert several extracellular proinflammatory activities. For example, S100A9 stimulates neutrophil adhesion to fibrinogen (19), while S100A8 has been reported to be an extremely potent chemotactic factor for murine myeloid cells (20). In addition, S100A8/A9 enhances migration of monocytes across endothelial cells (21). Endothelial cells incubated with S100A12 express increased levels of ICAM-1 and VCAM-1 due to an induction of NF-{kappa}B, resulting in the adhesion of lymphocytes to endothelial cells (22). In addition, S100A12 mediates expression of TNF-{alpha} and IL-1{beta} when incubated with a murine macrophage cell line (22).

Local secretion of MRPs has been detected in chronic periodontal infections (23, 24) and particularly high concentrations were found in the serum of patients suffering from chronic bronchitis, cystic fibrosis, and tuberculosis (7, 16, 25, 26). High levels of serum S100A8/A9 have also been found in HIV-1-seropositive patients with advanced immune deficiency (Centers for Disease Control and Prevention stages II and III) (27, 28, 29). Elevated S100A8/A9 levels in AIDS patients correlated with the onset of opportunistic infections. Other studies demonstrated high levels of S100A8/A9 in cerebrospinal fluids, saliva, and inflamed gingiva from AIDS patients during ongoing opportunistic infections (30, 31, 32). Some of these studies revealed that MRP levels were inversely proportional to CD4+ T cell counts and in linear correlation with viral load in patients with advanced HIV-1 disease (i.e., AIDS) (28, 29). More recently, S100A8 derived from cervico-vaginal secretions was shown to induce virus production in a latently HIV-1-infected monocytoid cell line (33). Despite the numerous data presenting an association between MRPs and HIV-1 pathogenesis, the understanding of this correlation remains elusive.

Regulation of HIV-1 is intimately linked to the activity of its promoter positioned in the 5' LTR sequence which is, in turn, dependent upon an enhancer region (-104/-81) necessary for a productive HIV-1 infection cycle in infected cells (34, 35, 36, 37). The enhancer region is characterized by two tandemly positioned NF-{kappa}B elements and, as demonstrated recently, a NFAT binding site (35, 38, 39, 40). Since then, it has been shown that the NF-{kappa}B and NFAT transcriptional elements act synergistically upon the viral enhancer region to positively modulate HIV-1 transcriptional activity and replication (40, 41, 42, 43, 44). Therefore, HIV-1 replication is tightly linked to T cell activation due to overlapping signal transduction requirements between cellular gene expression and activation of viral regulatory elements.

We set out to assess the possible modulatory effect of recombinant S100A8, S100A9, S100A12, and S100A8/A9 proteins on the life cycle of HIV-1 in a cell type recognized as a major cellular reservoir of this retrovirus, i.e., the CD4+ T cell. Using Jurkat-derived cell lines and freshly isolated PBMCs, we show in this study that MRPs induce HIV-1 transcriptional activity and virus gene expression through a NF-{kappa}B-dependent signal transduction pathway. These results suggest that MRPs should be considered as secreted factors contributing to the pathogenesis of HIV-1 infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells used in the current study

The lymphoid Jurkat T cell line (45) was obtained from the American Type Culture Collection (Manassas, VA). The 1G5 T cell line is a Jurkat derivative that harbors two stably integrated constructs constituted of the luciferase gene under the control of the HIV-1SF2 LTR (46). 1G5 cells were obtained through the National Institutes of Health AIDS Repository Reagent Program. We also made use of J1.1, a Jurkat E6.1-derived T cell line that is latently infected with HIV-1 (47). All of these cell lines were maintained in complete culture medium made of RPMI 1640 supplemented with 10% FBS (HyClone Laboratories, Logan, UT), glutamine (2 mM), penicillin G (100 U/ml), and streptomycin (100 µg/ml). Primary PBMCs from healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation. Before being used for preparation of nuclear extracts, these cells were first cultured in complete RPMI medium containing 20% FBS in the presence of 3 µg/ml of PHA-P (Sigma-Aldrich, St. Louis, MO) and 30 U/ml of recombinant human IL-2 for 3 days at 37°C under a 5% CO2.

Plasmids

The p{kappa}B-TATA-LUC plasmid containing the HIV-1 {kappa}B enhancer region (-105/-70) and a TATA box placed upstream of the luciferase reporter gene was used in this study (48). This plasmid was a generous gift from Dr. W. C. Greene (The J. Gladstone Institutes, San Francisco, CA). pNF-{kappa}B-LUC contains five consensus NF-{kappa}B-binding sequences placed upstream to the luciferase gene along with a minimal promoter (Stratagene, La Jolla, CA). The dominant negative I{kappa}B{alpha}-expressing vector pCMV-I{kappa}B{alpha}S32A/36A has been described previously (48) (a kind gift from Dr. W.C. Greene). The DNA filler pCMV-EcoRV/SmaI was constructed from the expressing vector pCMV-I{kappa}B{alpha} S32A/36A with EcoRV/SmaI digestion. pm{kappa}B-LTR-Luc was kindly provided by Dr. K. Calame (Columbia University, New York, NY). It contains the luciferase reporter gene under the control of mutated NF-{kappa}B (CTCACTTTCC) HIV-1 LTR (-453 to +80) (49). pNFAT-Luc, which contains the minimal IL-2 promoter with three tandem copies of the NFAT-binding site, was a kind gift from Dr. G. Crabtree (Howard Hughes Medical Institute, Stanford, CA).

rMRPs and polyclonal anti-MRP antiserum

Human S100A8, S100A9, and S100A12 cDNAs were synthesized by RT-PCR from neutrophil RNA isolated using TRIzol reagent according to the manufacturer’s instructions (Life Technologies, Grand Island, NY). cDNAs were cloned into the pET28 expression vector (Novagen, Madison, WI) and transformed in Escherichia coli HMS174. Expression of rMRPs was induced with 1 mM isopropyl {beta}-D-thiogalactoside for 16 h at 16°C. After incubation, cultures were centrifuged at 5,000 x g for 10 min. The pellet was resuspended in PBS/0.5 M NACl/1 mM imidazole and lysed by sonication. Lysates were then centrifuged at 55,000 x g for 25 min and the supernatants were collected. Recombinant His-tagged MRPs were purified using a nickel column. His-tagged proteins bound to the column were cleaved from their His-tag by adding 10 U of thrombin and were incubated for 16 h at room temperature. rMRPs were eluted with PBS. The digestion and elution process was repeated once to cleave the remaining undigested recombinant proteins. Contaminating thrombin was extracted from the eluates using streptavidin-agarose and contaminating LPS was removed by polymyxin B-agarose (Pierce, Rockford, IL). Eluted proteins were analyzed by immunoblot and SDS-PAGE. Control for bacterial contamination of the recombinant proteins consisted of untransformed HMS174 stimulated, lysed, and processed for purification like the recombinant proteins. Stimulation experiments with this control were conducted using the same dilutions as for MRPs. Proteins were inactivated by heating at 90°C for 15 min or by incubation with polyclonal Abs (anti-MRPs) for 30 min at room temperature before incubation with T lymphocytes. Polyclonal antisera against recombinant human S100A8, S100A9, and S100A12 were generated after repeated injections in New Zealand White rabbits at 4-wk intervals.

ELISA

The measurement of virus-encoded p24 protein was determined by an inhouse enzymatic assay and was described previously (50).

Transfections

Transient transfections of T cell lines using the DEAE-dextran method were performed as previously described (51). Briefly, cells (5 x 106) were first washed once in transfection solution buffer (137 mM NaCl, 25 mM Tris-HCl (pH 7.4), 5 mM KCl, 0.6 mM Na2HPO4, 0.5 mM MgCl2, and 0.7 mM CaCl2) and incubated in 0.5 ml of transfection solution containing 15 µg of DNA from the indicated plasmid(s) and 500 µg/ml of DEAE-dextran (final concentration; Pharmacia, Piscataway, NJ) for 25 min at room temperature. Thereafter, cells were diluted at a concentration of 1 x 106/ml using complete culture medium supplemented with 100 µM of chloroquine (Sigma-Aldrich) and transferred into 6-well plates. After 45 min of incubation at 37°C, cells were centrifuged, resuspended in complete culture medium, and incubated at 37°C for 24 h. To minimize variations in plasmid transfection efficiencies, cells were transfected in bulk and were next separated into various treatment groups.

Stimulations and reporter gene assays

Cells were seeded at a density of 105 cells per well (100 µl) in 96-well flat-bottom plates. Cells were either left unstimulated or treated with PMA (20 ng/ml; Sigma-Aldrich)/ionomycin (iono, 1 µM; Calbiochem, La Jolla, CA) or the indicated concentrations of MRPs in a final volume of 200 µl for 8 h at 37°C unless otherwise specified. Luciferase activity was then determined with a Dynex 96-well plate luminometer device (Chantilly, VA) following a previously described protocol (51).

Preparation of nuclear extracts and EMSA

Cells (5 x 106) were either left untreated or incubated for 1 h at 37°C with PMA/iono or MRPs (10 µg/ml). Incubation with the stimulating agents was terminated by the addition of ice-cold PBS and nuclear extracts were prepared according to the described microscale preparation protocol (52). Protein concentrations were determined by the bicinchoninic assay with a commercial protein reagent kit (Pierce). EMSA was performed with 10 µg of nuclear extracts incubated for 20 min at room temperature in 20 µl of 1x binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM DTT, 5 mM EDTA, 250 mM NaCl, 2 µg poly(dI-dC), 10 µg nuclease-free BSA fraction V) containing 0.8 ng of [{gamma}-32P]-labeled dsDNA oligonucleotide. The following consensus binding site dsDNA oligonucleotides were synthesized inhouse and used as probes and/or competitors: NF-{kappa}B (5'-ATGTGAGGGGACTTTCCCAG-GC-3'); Oct-2A (5'-GGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3'); and enhancer region (-104/-82) from the LTR of HIV-1 NL4-3 strain (5'-CAAGGGACTTTCCGCTGGGGACTTTCCAGGG-3'). DNA-protein complexes were resolved from free-labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels. The gels were subsequently dried and autoradiographed. Cold competition assays were conducted by adding a 100-fold molar excess of unlabeled dsDNA oligonucleotide simultaneously with the labeled probe. Supershift assays were performed by preincubation of nuclear extracts with 1 µg of Ab (anti-p50 subunit from NF-{kappa}B; Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of all the components of the binding reaction for 30 min on ice before the addition of the labeled probe.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MRPs promote virus production in a latently HIV-1-infected T cell line

To decipher the possible modulatory role of MRPs on virus production, a latently HIV-1-infected established T lymphoid cell line (i.e., J1.1) was treated with S100A8, S100A9, S100A12, or S100A8/A9. As depicted in Fig. 1Go, the level of virus production was strongly up-regulated in J1.1 cells by all MRPs tested by an ~4-fold induction when compared with untreated cells. No such induction of HIV-1 production could be seen when J1.1 cells were instead treated with mock protein purification solution, therefore demonstrating that the noticed enhancing effect was not due to a bacterial product contaminant. These results indicate that MRPs are potent inducers of HIV-1 production in human CD4+ T cells. These observations prompted us to investigate at the molecular level the different intracellular events leading to MRP-mediated activation of HIV-1 gene expression in CD4-expressing T cells.



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  		FIGURE 1. Treatment of latently HIV-1-infected T cells with MRPs lead to virus production. J1.1 cells were either left untreated or were treated with the mock protein purification solution (using the same dilution as for MRPs) or 10 µg/ml of MRPs (i.e., S100A8, S100A9, S100A12, and S100A8/A9) for 24 h at 37°C. Supernatants were harvested and virus production was evaluated using a p24 enzymatic assay. Results shown are the means ± SEM of triplicate samples from one experiment that is representative of two others.

 
MRPs mediate activation of HIV-1 transcription

In an attempt to study the effect of MRPs on HIV-1 LTR-driven transcriptional activity, both a dose response and kinetic analyses were conducted using the Jurkat-derived 1G5 T cell line, which carries two stably integrated constructs made of the luciferase reporter gene driven by the HIV-1SF2 LTR. As shown in Fig. 2GoA, a significant up-regulation of HIV-1 LTR activity was observed upon treatment of 1G5 cells with concentrations as low as 1 µg/ml of S100A8, S100A9, S100A12, or S100A8/A9. Maximal luciferase activity was observed when using MRPs at a concentration of 10 µg/ml. This protein concentration (10 µg/ml) was thus used throughout the following sets of experiments unless specified otherwise. It should be noted that all MRPs tested were able to activate HIV-1 LTR-dependent reporter gene activity.



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  		FIGURE 2. Dose response and kinetic analysis of MRP-mediated induction of HIV-1 LTR activity. A, 1G5 cells were either left untreated of were treated with the mock protein purification solution (using the same dilution as for MRPs), PMA/iono (20 ng/ml and 1 µM, respectively), or increasing doses of MRPs (S100A8, S100A9, S100A12, and S100A8/A9) ranging from 0.01 to 50 µg/ml for 8 h at 37°C. B, 1G5 cells were either left untreated or were treated with the mock protein purification solution, PMA/iono, or MRPs (S100A8, S100A9, S100A12, and S100A8/A9) (10 µg/ml) for the indicated time periods. Cells were then lysed and luciferase activity was monitored with a microplate luminometer. Results are presented as fold induction in luciferase activity either over untreated samples for PMA/iono-treated samples or over mock protein purification solution-treated samples from MRP-treated samples. These data are from the calculated means ± SEM of three different lysed cell samples in the same experimental setting and are representative of three different experiments.

 
Kinetic analysis of HIV-1 LTR activity was also performed using 1G5 cells stimulated with MRPs for increasing periods of time. As shown in Fig. 2GoB, a significant increase in HIV-1 LTR activity was detected as early as 2 h following stimulation whereas maximal activation was observed ~8-h posttreatment. PMA/iono stimulation, used as a positive control, resulted in a 69-fold induction over untreated control. These results demonstrate that the effect of MRPs on HIV-1 LTR activity is dose-dependent and transient in human CD4+ T cells.

To demonstrate the specificity of MRP-mediated HIV-1 LTR activity, we performed a set of experiments in which MRPs were either denaturated by heat or inhibited using MRP-specific neutralizing antisera. As shown in Fig. 3GoA, physical denaturation of these proteins completely abolished their ability to induce LTR activation in 1G5 cells. To further address and confirm the specificity of the MRP-mediated effect on HIV-1 LTR activity, we made use of neutralizing polyclonal antisera directed against MRPs. As shown in Fig. 3GoB, the addition of specific Abs to the MRPs significantly diminished MRP-mediated luciferase activity in 1G5 cells when compared with preimmune sera. In addition, the antisera were ineffective at inhibiting PMA/iono-induced LTR activity, thus confirming that the effect detected was caused by the MRPs.



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  		FIGURE 3. MRP-mediated induction of HIV-1 LTR-driven activity is abolished by heat denaturation and MRP-specific Abs. A, 1G5 cells were treated with the mock protein purification solution, PMA/iono combination, or with the native or heat inactivated MRPs (i.e. S100A8, S100A9, S100A12, and S100A8/A9) (10 µg/ml). B, 1G5 cells were treated with the mock protein purification solution, PMA/iono, or MRPs (5 µg/ml) and incubated with either MRP-specific polyclonal Abs or preimmunized control serum. Cells were then lysed and luciferase activity was monitored using a microplate luminometer. Results are presented as fold induction in luciferase activity either over untreated samples for PMA/iono-treated samples or over mock protein purification solution-treated samples form MRP-treated samples. These data are from the calculated means ± SEM of three different lysed cell samples in the same experimental setting and are representative of three different experiments.

 
MRPs are acting on HIV-1 enhancer region

Because it is well-established that HIV-1 transcriptional activity and expression is predominantly driven through the LTR enhancer domain (-104/-82), we set out to evaluate whether MRP-induced HIV-1 activity acts through the HIV-1 enhancer using a Jurkat cell line transiently transfected with the luciferase gene under the control of the LTR enhancer region (i.e., p{kappa}B-TATA-Luc). These cells were either left untreated or stimulated with PMA/iono or MRPs. As shown in Fig. 4GoA, all MRPs significantly up-regulated the LTR enhancer-driven luciferase activity in Jurkat cells. In an attempt to confirm these observations, EMSA were performed in both Jurkat cells and freshly isolated human PBMCs. Nuclear extracts from untreated, PMA/iono-, S100A8-, S100A9-, or mock protein purification solution-treated Jurkat cells or PBMCs were incubated with radiolabeled probes corresponding to the HIV-1 LTR enhancer sequence. As illustrated in Fig. 4GoB, nuclear extracts from cells treated with PMA/iono (lane 2) or MRPs (lanes 3 and 4) demonstrated the presence of intense enhancer/protein complexes when compared with complexes obtained from untreated (lane 1) or mock protein purification solution-treated cells (lane 5). Specific and nonspecific competitions using 100-fold concentrations of nonlabeled probes confirmed the specificity of the signal (lanes 6 and 7). Altogether, these results strongly suggest that the enhancer domain is playing a key role in the MRP-mediated effect on HIV-1 LTR activity.



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  		FIGURE 4. MRPs mediate activation of HIV-1 enhancer and lead to the binding of cellular factors onto the enhancer domain. A, Jurkat cells were transiently transfected with an HIV-1 enhancer-driven luciferase reporter gene construct, i.e., p{kappa}B-TATA-Luc. Cells were treated with the mock protein purification solution, PMA/iono, or MRPs (10 µg/ml) for 8 h at 37°C, then lysed and luciferase activity was monitored using a microplate luminometer. Results are presented as fold induction in luciferase activity either over untreated samples for PMA/iono-treated samples or over mock protein purification solution-treated samples form MRP-treated samples. These data are from the calculated means ± SEM of three different lysed cell samples in the same experimental setting and are representative of three different experiments. B, Labeled HIV-1 enhancer oligonucleotide was incubated with nuclear extracts from Jurkat cells or PBMCs that were either left untreated (lane 1) or were treated with PMA/iono combination (lane 2), S100A8 (lane 3), S100A9 (lane 4), or the mock protein purification solution (lane 5). Binding specificity was tested by adding a 100-fold molar excess of a probe composed of either the cognate HIV-1 enhancer oligonucleotide (lane 6) or a nonspecific probe (i.e., Oct-2A) (lane 7). These results are from one experiment that is representative of two others.

 
HIV-1 activation induced by MRPs is mediated through the NF-{kappa}B factor

Previous findings demonstrated that NF-{kappa}B and NFAT can act in a concerted manner to up-regulate the HIV-1 enhancer region (43, 53). To delineate the involvement of these two transcription factors in MRP-mediated HIV-1 activation, we transiently transfected Jurkat cells with a luciferase-based reporter vector that is regulated by several NF-{kappa}B consensus binding sites. Data from Fig. 5GoA demonstrate that all MRPs tested act as strong inducers of NF-{kappa}B. This observation suggests that the transcriptional factor NF-{kappa}B is responsible for the MRP-dependent up-regulation of HIV-1 gene expression. In contrast, MRPs were unable to mediate activation of NFAT as assessed by transient transfection of Jurkat cells with a NFAT-driven reporter gene construct (data not shown). To substantiate the involvement of NF-{kappa}B in the observed MRP-mediated induction of HIV-1 LTR activity, Jurkat cells were next transiently transfected with an HIV-1 LTR-driven reporter gene vector that carries mutations in the two NF-{kappa}B binding sites. When using this molecular construct, MRPs were no longer able to mediate activation of the regulating elements of HIV-1 (Fig. 5GoB), thereby confirming the pivotal role played by NF-{kappa}B in this phenomenon.



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  		FIGURE 5. MRPs mediate NF-{kappa}B-dependent up-regulation of HIV-1 LTR activity. Jurkat cells were first transiently transfected with pNF-{kappa}B-Luc (A), pLTR-Luc (B), or pm{kappa}B-TATA-Luc (B). Cells were next treated with either the mock protein purification solution, PMA/iono, or MRPs (10 µg/ml) for 8 h at 37°C. Cells were then lysed and monitored for luciferase activity. Results are presented as fold induction in luciferase activity over either untreated samples for PMA/iono-treated samples or over mock protein purification solution-treated samples from MRP-treated samples. These data are from the calculated means ± SEM of three different lysed cell samples in the same experimental setting and are representative of three different experiments.

 
Our next series of investigations were aimed at defining the nature of the protein complex that binds to the HIV-1 enhancer following treatment with MRPs. To this end, mobility shift assays were conducted using again as a probe the HIV-1 enhancer region that was incubated along with nuclear extracts from S100A8-treated Jurkat cells and human PBMCs. A more intense retarded complex was detected following treatment of Jurkat cells and PBMCs with either PMA/iono (positive control) or S100A8 (Fig. 6Go, compare lanes 2 and 3 with lane 1). The formation of the complex induced by S100A8 treatment was totally competed away by an excess of unlabeled consensus NF-{kappa}B probe (lane 4) but not NFAT and Oct-2A probes (lanes 6 and 9). Finally, the S100A8-induced complex was supershifted when anti-p50 polyclonal Abs were included in the EMSA reaction mixture (lane 5) demonstrating that the p50 subunit of NF-{kappa}B is a major component of the retarded complex that binds to the HIV-1 enhancer region.



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  		FIGURE 6. NF-{kappa}B is the predominant factor binding to the LTR enhancer sequence in CD4+ T cells upon MRP-induced activation. Labeled HIV-1 enhancer oligonucleotides were incubated with nuclear extracts from Jurkat cells or PBMCs that were either left untreated (lane 1) or were treated with PMA/iono combination (lanes 2 and 8), or S100A8 (lane 3–7 and 9) for 1 h at 37°C. Binding specificity was tested by adding a 100-fold molar excess of a probe composed of either the consensus NF-{kappa}B binding site (lane 4), the cognate HIV-1 enhancer oligonucleotide (lanes 7 and 8), or a nonspecific probe (i.e., Oct-2A) (lane 9). For gel supershift assays, nuclear extracts were also incubated with an Ab specific for the p50 subunit of NF-{kappa}B (lane 5). These results are from one experiment that is representative of two others. NA, not applicable.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Opportunistic infections frequently result in an increase in viral load in HIV-1-infected individuals and a decrease in CD4+ T cell count. A number of inflammatory mediators released in the serum of the patients during these pathologies contribute to HIV-1 replication (1, 2, 3). Recently, cytokine-like activities have been ascribed to MRPs (19, 21, 22), which are released during inflammatory episodes by neutrophils and monocytes (54, 55, 56, 57). High levels of MRPs have been detected in the serum of patients suffering from bronchitis and tuberculosis (7, 16, 26), as well as in patients suffering from AIDS (27, 28, 29, 30, 31, 32). The presence of these proteins in HIV-1 seropositive patients was correlated with an increased viral load and a decrease in the number of circulating CD4-expressing T lymphocytes. Because MRPs have been recently demonstrated to activate monocytes as well as lymphocytes, it was tempting to speculate that the release of MRPs during opportunistic infections could affect the replicative cycle of HIV-1 in T lymphocytes.

The data presented in this study demonstrate that MRPs up-regulate HIV-1 transcriptional activity and virus production in CD4+ T lymphocytes. We show that recombinant S100A8, S100A9, S100A12, and S100A8/A9 proteins induce HIV-1 replication in a chronically infected Jurkat cell line (J1.1). More specifically, MRP treatment resulted in a dose- and time-dependent activation of the HIV-1 LTR promoter sequence that is exerted through the enhancer region. Maximal stimulation with MRPs occurred at 10 µg/ml, a concentration previously demonstrated to be in the range of those found in the serum of HIV-1-infected patients (14 µg/ml) (27), thereby providing a physiological relevance to the present findings. Although S100A8 and S100A12 are chemotactic at much lower concentrations (10 ng/ml), similar concentrations had been reported to induce VCAM-1 and ICAM-1 expression by endothelial cells, and IL-2 production by PBMCs (21, 22). Mechanistic analysis revealed that MRP-induced HIV-1 activation was dependent upon the NF-{kappa}B binding sites located within the HIV-1 LTR enhancer region.

Several known inducers of HIV-1 transcription such as PGE2, TNF-{alpha}, anti-CD3 Abs, and phorbol ester agents (e.g., PMA) promote HIV-1 replication through an induction of the transcription factor NF-{kappa}B (39, 58). NF-{kappa}B is also important for expression of several cytokines (e.g., IL-1, IL-6, and TNF-{alpha}) and adhesion molecules (e.g., ICAM-1 and VCAM-1) (59, 60, 61). The fact that inflammatory proteins such as MRPs activate HIV-1 replication in CD4+ T cells in a NF-{kappa}B-dependent manner is in agreement with the previous observations indicating that S100A12 mediates nuclear translocation and activation of NF-{kappa}B (22). In this work, activation of NF-{kappa}B by S100A12 resulted in expression of ICAM-1 and VCAM-1 by endothelial cells. Therefore, interaction between MRPs and T cells is likely to result not only in HIV-1 gene expression and virus production, as shown in the present study, but also in an increased surface expression of ICAM-1 on such a cellular subpopulation. Given that ICAM-1 has been shown to be inserted within the HIV-1 envelope and to result in an enhancement of virus infectivity (50, 62), production of MRPs due to some specific opportunistic infections could also result in production of HIV-1 particles that would be more infectious.

In addition to ICAM-1, S100A12 also induces IL-2 production and secretion from PBMCs, as well as the release of TNF-{alpha} and IL-1{beta} from mononuclear phagocytes (22). It can thus be proposed that such a MRP-mediated release of cytokines can also enhance viral load by directly augmenting HIV-1 production. The kinetic of activation of HIV-1 LTR-driven transcriptional activity, with a peak reached at 6–8 h following treatment, represents an indication that the observed up-regulating effect of MRPs is direct and not via the production of proinflammatory cytokines that could act in an autocrine/paracrine fashion to activate virus production. This kinetic is similar to the ones reported for PMA and TNF-{alpha} which directly act on the HIV-1 promoter (58). In addition, mobility shift assays, using nuclear extracts from MRP-treated Jurkat and PBMCs, demonstrate a rapid translocation of NF-{kappa}B following cellular activation (i.e., 60-min posttreatment) confirming a direct action of MRPs on the HIV-1 LTR. Nevertheless, the possibility that MRPs could lead to the production of TNF-{alpha} and/or IL-1{beta} in T lymphocytes, which would in turn activate LTR activity, cannot be completely ruled out.

The transcription factor NF-{kappa}B was found to play an essential role in MRP-dependent activation of HIV-1 gene expression, while NFAT played no role at all. It is possible that NFAT would play a more important role with respect to MRP-mediated induction of HIV-1 LTR activity in cells such as naive T cells that are known to express higher levels of NFAT compared with NF-{kappa}B (63). Alternatively, cytokines such as IL-2, a potent activator of NFAT, might act synergistically with MRPs to induce HIV-1 transcription. Further studies are required to solve this issue.

The results presented in this study reveal for the first time that MRPs such as S100A8, S100A9, S100A12, and S100A8/A9 can up-regulate HIV-1 transcription and virus production in human CD4-expressing T cells. Although we have been able to define that MRPs mediate their positive effect on HIV-1 LTR via an NF-{kappa}B signaling pathway, the cellular receptor(s) that bind MRPs on human CD4+ T cells has not been investigated. The receptor for advanced glycation end-products (RAGE) was recently reported to be a natural receptor for S100A12; to date, RAGE remains the sole receptor known to bind MRPs. However, S100A8 and S100A9 do not bind to RAGE-transfected Chinese hamster ovary cells, suggesting that they interact with a distinct receptor (64). Although we observed RAGE gene expression in PBMCs using RT-PCR (data not shown), we suspect that a distinct receptor is responsible for the MRP-mediated induction of NF-{kappa}B in CD4+ lymphocytes. Inhibition of RAGE with specific Abs or by competition with soluble RAGE could confirm this hypothesis.

Previous studies have indicated that high levels of S100A8/A9 are found in sera from HIV-1-infected patients, which correlated with the evolution of immune deficiency and opportunistic infections (27, 28, 29). Whether the presence of MRPs in the serum of these patients is a consequence or a cause of HIV-1 replication remains unknown. Interestingly, Hashemi et al. (33) demonstrated recently that S100A8 protein derived from cervico-vaginal secretions activates HIV-1 replication in a latently infected monocytoid cell line. This observation, along with the data presented in this study, could help to explain the correlation between the increased levels of serum MRPs and the reduction of CD4+ cell counts and enhancement of HIV-1 load (28, 29). Globally, our results suggest that MRPs could represent significant contributors to HIV-1 disease progression in seropositive patients with ongoing opportunistic infections and/or inflammatory conditions.


   Acknowledgments
 
We thank Pascal Rouleau for his technical help in the purification of the recombinant MRPs.


   Footnotes
 
1 This study was supported by an Arthritis Society of Canada Grant (to P.A.T.) and Canadian Institutes of Health Research (CIHR) HIV/AIDS Research Program Grants HOP-14438, MOP-37781, and HOP-15575 (to M.J.T.). C.R. is supported by a studentship from the K.H. Hunter Charitable Foundation and the CIHR. J.R. is the recipient of a CIHR Doctoral Award, while G.A.R. holds a PhD Fellowship from the Fonds de la Recherche en Santé du Québec-Fonds pour la Formation de Chercheurs et l’Aide à la Recherche Health Program. P.A.T. holds a Scholarship Award from the Arthritis Society of Canada and M.J.T. has been allocated a Tier 1 Canada Research Chair in Human Immunoretrovirology. Back

2 C.R., G.A.R., and J.R. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Philippe A. Tessier and Dr. Michel J. Tremblay, Centre de Recherche en Infectiologie, Local RC709, Pavillon Centre Hospitalier de l’Université Laval, Centre Hospitalier Universitaire de Québec, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail addresses: Philippe.Tessier@crchul.ulaval.ca and Michel.J.Tremblay{at}crchul.ulaval.ca Back

4 Abbreviations used in this paper: HIV-1, HIV type 1; LTR, long terminal repeat; MRP, myeloid-related protein; iono, ionomycin; RAGE, receptor for advanced glycation end-product. Back

Received for publication May 20, 2002. Accepted for publication July 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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