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The Journal of Immunology, 2001, 166: 4244-4253.
Copyright © 2001 by The American Association of Immunologists

Opposite Effects of IL-10 on the Ability of Dendritic Cells and Macrophages to Replicate Primary CXCR4-Dependent HIV-1 Strains1

Petronela Ancuta2,*, Youssef Bakri{dagger},{ddagger}, Nicolas Chomont*, Hakim Hocini*, Dana Gabuzda§ and Nicole Haeffner-Cavaillon*

* Unité d’Immunopathologie Humaine, Institut National de la Santé et de la Recherche Médicale, Broussais Hospital, Paris, France; {dagger} Institut National de la Santé et de la Recherche Médicale E0013, Faculté de Médicine Saint-Antoine, Paris, France; {ddagger} Laboratoire de Biochimie-Immunologie, JER 3012 associée à l’AUPELF, Faculté de Sciences, Rabat, Morocco; and § Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (M{Phi}s). M{Phi}s efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by M{Phi}s as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and M{Phi}s, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in M{Phi}s. The effect of IL-10 on the ability of DCs and M{Phi}s to transmit virus to autologous CD4+ T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did M{Phi}s to transmit a vigorous infection to CD4+ T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in M{Phi}:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and M{Phi}s. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4+ T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Strains of HIV-1 isolated from recently infected individuals are predominantly macrophage (M{Phi})3-tropic, nonsyncitium inducing, and use CCR5 as a coreceptor in combination with CD4 (1). Later in the course of HIV-1 disease, the virus frequently expands its coreceptor use to include CXC chemokine receptor (CXCR) 4, CCR3, CCR2, and other coreceptors in addition to CCR5, resulting in a selective advantage for the virus (2). The emergence of CXCR4-dependent HIV-1 (X4 HIV-1) strains precedes a more rapid decline in CD4+ T cell counts and progression to AIDS, suggesting that X4 strains contribute to AIDS pathogenesis (3). Furthermore, an early acquisition of CXCR4 usage predicts a poor prognosis (1, 4). Because the rate of HIV turnover in vivo is high (5, 6) and a small number of amino acid mutations in gp160 envelope glycoprotein is sufficient to change viral tropism (7, 8), it is not clear why CXCR4-dependent viruses do not emerge earlier.

The high levels of viremia at advanced stages of HIV-1 disease when CD4+ T cells are markedly depleted suggests that other cells may be responsible for viral replication in vivo. After several controversial reports, it is now established that dendritic cells (DCs) and M{Phi}s play an important role in the natural history of HIV-1 infection. DCs and M{Phi}s are assumed to be the first targets of viral infection in the genital or rectal mucosa (9, 10) and serve as long-term reservoirs in chronically HIV-1-infected patients (11, 12, 13, 14, 15). Tissue M{Phi}s represent a highly productive source of HIV during opportunistic infections at advanced stages of HIV disease (15), whereas DCs exhibit a high capacity to transmit virus to CD4+ T lymphocytes, even in the absence of DC infection per se (16, 17, 18, 19). DCs and M{Phi}s express CCR5 and are highly susceptible to infection by CCR5-dependent HIV-1 (R5 HIV-1) strains (16, 20, 21, 22, 23). DCs and M{Phi}s also express functional CXCR4 HIV-1 coreceptor and support replication of primary X4 HIV-1 strains (24, 25). However, laboratory-adapted X4 HIV-1 strains such as IIIB appear to be blocked during postentry steps in M{Phi}s (26). Interestingly, exposure to bacterial products renders M{Phi}s highly susceptible to X4 HIV-1 strains in vitro (27). Similarly, it has been reported that maturation of DCs is associated with up-regulation of CXCR4 expression and a higher susceptibility of DC to infection with X4 HIV-1 strains (28, 29). These observations raise the possibility that DCs and M{Phi}s may be involved in promoting the transition from an R5 to X4 HIV-1 phenotype during the natural course of HIV infection.

Several studies have reported a Th1/Th2 shift in the cytokine pattern from production of IL-2/IFN-{gamma} toward IL-4/IL-10 during the course of HIV-1 infection (30). A shift from R5 to X4 HIV-1 variants has also been associated with disease progression (1). Based on these observations, it has been suggested that Th2 cytokines may have an important role in the evolution of HIV-1 tropism (31). The cytokine environment may determine susceptibility to HIV infection in part by the regulation of HIV-1 coreceptor expression (32). IL-4 down-regulates CCR5 but up-regulates CXCR4 expression on CD4+ T cells and favors high replication of X4 HIV-1 strains (33). IL-4 can also increase HIV-1 replication in DC:T cell cocultures by stimulating T cell proliferation (34). IL-4-treatment renders DCs highly susceptible to infection with X4 HIV-1 strains because of CXCR4 up-regulation (25). In contrast, IL-4 diminishes CCR5 expression and replication of R5 HIV-1 strains by M{Phi}s (32, 35). These observations suggest that IL-4 may play a role in the switch of HIV-1 strains from an R5 to X4 phenotype in vivo (36).

Increased levels of IL-10 in serum of HIV-1-infected patients have been reported to coincide with a dramatic decrease in CD4+ T cells counts and progression to AIDS (37, 38). The role of IL-10 in the emergence of X4 HIV-1 strains in vivo is unknown. It has been reported that IL-10 up-regulates CCR5 expression on monocyte (Mo)/M{Phi} but blocks R5 HIV-1 replication at a postentry step (32). In contrast, IL-10 decreases both CCR5 expression and R5 HIV-1 strain replication in CD4+ T lymphocytes (39). Studies on the effect of IL-10 on CXCR4 expression on CD4+ T lymphocytes have reported contradictory results (39, 40).

In view of the coincidence between the emergence of X4 HIV-1 strains and the high levels of IL-10 in the serum of patients at advanced stages of HIV-1 disease, we investigated the effect of IL-10 on the capacity of Mo-derived M{Phi}s and DCs to replicate primary X4 HIV-1 strains. We also examined the effect of IL-10 on X4 HIV-1 replication in M{Phi}/DC and T cell cocultures, which more closely mimic the microenvironment of lymphoid tissues. In this study, we show that IL-10 inhibits replication of X4 HIV-1 strains in M{Phi}s but significantly increases viral replication in DCs. We also show that IL-10 decreases X4 HIV-1 replication in M{Phi}:T cell cocultures, but has no inhibitory effect on viral replication in DC:T cell cocultures. These results suggest that IL-10 may favor the emergence of X4 HIV-1 strains at advanced stages of HIV disease.


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

Human rM-CSF, GM-CSF, IL-4, and IL-10 were purchased from R&D System Europe (Oxon, U.K.). Anti-CD14 (My4)-FITC, anti-CD16-PECy5, anti-CD11b-PE, anti-CD80-FITC, anti-CD86-FITC, anti-CD83-PE mAbs were from Immunotech (Beckman Coulter, Villepinte, France); anti-CCR5-PE, anti-CXCR4-PE anti-HLA-DR-FITC, anti-CD33-PE, anti-CD4-PE, and anti-CD3-PE mAb were from BD Becton Dickinson (Le Pont de Claix, France). Anti-CD40-FITC mAb was from Calbiochem-Novabiochem (San Diego, CA). Anti-CD1a-FITC mAbs were purchased from Tebu (Le Perray, France).

Purification of CD4+ T lymphocytes

PBMCs were isolated from the buffy coats of healthy donors by Ficoll gradient (Eurobio, Les Ulis, France). T lymphocytes were isolated from PBMC by E-rosette formation using 2-aminoethylisothiouronium bromide hydrobromide-treated SRBC. The rosetting T cells (E+) were separated from nonrosetting cells by gradient density. After lysis of SRBC with 0.83% NH4Cl, cells were washed twice in PBS and once in RPMI 1640 medium and suspended in culture medium. E+ cells contained >95% CD3+ cells as assessed by flow cytometry. T lymphocytes were then depleted of CD8+ T cells by using Dynabeads M-450 CD8 (Dynal France SA, Compiègne, France).

Isolation of Mo-derived M{Phi}s and DCs

T cell-depleted PBMCs (E-) were used for Mo isolation. The percentage of Mos in E- cells was determined by flow cytometry analysis and cell suspensions in RPMI 1640 medium containing 10% Ab human serum were cultured into 12-well culture plates (Costar, Cambridge, MA) at a concentration of 106 Mo/ml/well. After 1 h of incubation at 37°C, nonadherent cells were removed by several washings and the adherent cells (>95% Mos with a CD33+CD14+ phenotype) were further cultured in RPMI 1640 medium containing 10% FCS and appropriate human recombinant cytokines. Cell preparations did not contain CD3+ T lymphocytes, as determined by flow cytometry (data not shown). M{Phi}s and DCs were obtained by culturing Mos in the presence of M-CSF (10 ng/ml/106 cells) (41) and GM-CSF (10 ng/ml/106 cells) plus IL-4 (10 ng/ml/106 cells), respectively (42). In each experiment, M{Phi}s and DCs were obtained by differentiation of purified Mos from the same donor. To study the effect of IL-10 on Mos differentiation into M{Phi}s and DCs, we used a concentration of IL-10 (10 ng/ml/106 cells) that was shown to inhibit HIV-1Ba-L replication in M{Phi}s (unpublished data) (43). Every 2 days, 50% of medium was removed from each well and replaced by fresh medium containing the appropriate cytokines. At day 6, analysis of phenotype and HIV-1 replication was performed.

Flow cytometry analysis

The expression of membrane Ags by the different cell subsets was analyzed by flow cytometry using three-color direct immunofluorescence. After incubation with the different mAbs for 30 min at 4°C, cells were washed twice with PBS containing azide (0.01%), BSA (0.2%), and fixed using a 1% formaldehyde PBS buffer. An aliquot of cells fixed with a 4% formaldehyde PBS buffer was subsequently stained with fluorescent anti-CXCR4 mAbs in the presence of a saponin buffer. Stained cells were analyzed using a FACSCalibur flow cytometer (BD Becton Dickinson) and the CellQuest software. Subsequent gating according to light-scattering properties and CD33 expression permitted us to identify Mo-derived cells. Ten thousand events were collected in list mode files for each test. Fluorescence parameters were collected using a four-decade logarithmic amplification. The marker for positive cells was located according to isotype-matched mAbs in each cell subset.

Detection of CXCR4 mRNA

RT-PCR was performed using a modified RT-PCR protocol previously described by Trujillo et al. (44). Total RNA was isolated using RNeasy mini-kit (Qiagen, Courtaboeuf, France), primed (1 µg) with anti-sense CXCR4 or GAPDH primers, and reverse transcribed into cDNA in a 20-µl reaction mixture containing Superscript II reverse transcriptase and RNaseOUT rRNase Inhibitor (Life Technologies, Cergy Pontoise, France). A series of increasing amounts of cDNA (5 µl, 2.5 µl, and 1 µl) from the cDNA reaction mixture were subjected to PCR amplification using Taq-polymerase (Roche Diagnostic Systems, Meylon, France) in an automated DNA Thermal Cycler (Crocodile III; Appligene, Strasbourg, France) for 32 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 40 s (CXCR4), or at 55°C for 60 s (GAPDH), and extension at 72°C for 90 s, followed by a final extension at 72°C for 15 min. The following primers were used for CXCR4: 5'-GTT ACC ATG GAG GGG ATC A-3' and 5'-CAG ATG AAT GTC CAC CTC GC-3'; and for GAPDH 5'-GTG AAG GTC GGA GTC AAC G-3' and 5'-GGT GAA GAC GCC AGT GGA CTC-3'. RT-PCR products (15 µl) were visualized under UV transillumination by ethidium bromide staining after electrophoresis on a 2% agarose gel.

HIV strains and infections

X4 HIV-1NDK and HIV-1VN44 strains (45, 46) were kindly provided by Dr. Françoise Barré-Sinoussi (Pasteur Institute, France). After 6 days of differentiation, M{Phi}s and DCs obtained in the presence or absence of IL-10 were incubated with 4 or 20 ng/ml/106 of DNase-treated viral suspension for 12 h. Heat-inactivated virus (1 h at 56°C) was used as a negative control. Cells were then washed three times, counted, and further cultured in RPMI 1640 medium 10% FCS at 0.2 x 106 cells/ml/well into 24-well culture plates in the presence of appropriate cytokines (M-CSF, M-CSF plus IL-10, GM-CSF plus IL-4, or GM-CSF plus IL-4 plus IL-10). In parallel experiments, HIV-1NDK-infected M{Phi}s, M{Phi}/IL-10s, DCs, and DC/IL-10s were cocultured with autologous CD4+ T cells. Before cocultures, CD4+ T lymphocytes were stimulated with PHA (0.5 µg/ml) plus IL-2 (10 U/ml) for 3 days. Cocultures were performed using a M{Phi}/DC:T cell ratio of 1:1. In control experiments, PHA/IL-2-stimulated CD4+ T cells were directly infected with HIV-1NDK (20 ng/ml/106 cells). After 3 h, T cells were washed and further cultured at 0.2 x 106 cells/ml into 24-well culture plates in medium in the presence or absence of IL-10 or IL-10 plus IL-4 (10 ng/ml). Each sample was performed in duplicate. Culture supernatants were harvested every 3 days and appropriate cytokines were then added together with fresh medium. The kinetics of viral production was followed by sequential measurement of p24 in duplicate supernatants using the HIV-1 p24 core profile ELISA kit (NEN Life Sciences Products, France).

Detection of HIV-1 DNA

HIV-1 DNA was detected by nested PCR as described (29). Briefly, M{Phi}s and DCs were exposed to 4 or 20 ng/ml/106 cells HIV-1NDK or HIV-1VN44 and analyzed for the expression of Pol HIV-1 DNA products 24 h later. Cells were then washed and lysed in a buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 0.5% Tween 20 (Bio-Rad, Hercules, CA), 0.5% Nonidet (Sigma, St. Louis, MO) and proteinase K (20 µg/ml) (Boehringer, Mannheim, Germany). Cell lysates (106cells/ml) were incubated for 1 h at 56°C, and proteinase K was then inactivated at 90°C for 10 min. Relative amounts of virus in different samples were estimated by endpoint dilutions of the lysates in lysed HIV- CEM cells (1 x 106/ml). Serially diluted samples (30 µl) were added to 0.5 µmol/L of each primer, 0,25 µmol/L dNTP (Roche Diagnostic Systems), 1.5 mmol/L MgCl2, and 1 U Taq DNA polymerase (Roche Diagnostic Systems) in 50 µl final volume. After 5 min at 94°C, 35 cycles were performed in an automated DNA Thermal Cycler (Crocodile III; Appligene), each cycle consisting of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. Pol primers were P3 (5'-TGG GAA GTT CAA TTA GGA ATA CCA C-3') and P4 (5'-CCT ACA TAC AAA TCA TCC ATG TAT T-3') (47) (Perkin-Elmer, Foster City, CA). For the nested PCR, 2 µl of amplified products were submitted to another 35 cycles of amplification under the same conditions using internal primers P5 (5'-ATC AGT AAC AGT ACT GGA TGT G-3') and P6 (5'-GAT AGA TAA CTA TGT CTG GAT T-3') (Perkin-Elmer). PCR sensitivity was 1 copy/3 x 104 cells as determined relative to serial dilutions of 8E5/LAV cells (1 copy HIV/cell) in HIV- CEM cells. The approximate number of DNA copies was determined relative to the standard curve obtained with 8E5 cells. PCR was also performed with {beta}-globin primers PCO4 (5'-CAC TTC ATC CAC GTT CAC C-3') and GH2O (5'-GAA GAG CCA AGG ACA GGT AC-3') (Perkin-Elmer) as amplification and DNA content controls. Nested PCR products (15 µl) were visualized under UV transillumination by ethidium bromide staining after electrophoresis on a 2% agarose gel.

RANTES production

RANTES production by M{Phi}s, M{Phi}/IL-10s, DCs, and DC/IL-10s was quantitated in supernatants from HIV-1NDK-infected cells (2 x 105 cells/ml) at day 3 postinfection by ELISA (Medgenix Diagnostic, Fleurus, Belgium).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IL-10 on Mo differentiation into M{Phi}s or DCs

Mos were differentiated into M{Phi}s and DCs in the presence or absence of IL-10. At day 6, the expression of cell surface molecules specific for Mo/M{Phi} (CD14 and CD16) and for DC (CD1a and CD83) lineage as well as molecules involved in Ag presentation (CD40, CD80, CD86, HLA-DR) and phagocytosis (CD11b, CD11c) was assessed by flow cytometry analysis (Table IGo).


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  		Table I. Phenotypic characterization of M{Phi} and DC obtained in the presence or absence of IL-101

 
Cells cultured in the presence of M-CSF (M{Phi}s) were adherent and exhibited typical M{Phi} morphology (Fig. 1GoA). M{Phi}s expressed high levels of CD14, CD16, CD11b, and CD11c, low levels of CD40, HLA-DR, and a low percentage expressed CD80, CD86, and CD1a molecules (Table IGo). As expected, M{Phi}s were CD83- (Table IGo). Mo differentiated in the presence of M-CSF plus IL-10 (M{Phi}/IL-10) were adherent and exhibited a morphology similar to that of M{Phi}s (Fig. 1GoB). Phenotypic analysis indicated that M{Phi}/IL-10 did not express CD80, CD86, CD1a, and CD83 markers but expressed similar levels of CD14, CD16, HLA-DR, and CD11c, and lower levels of CD11b and CD40, compared with M{Phi}s (Table IGo). When Mos were cultured in the presence of GM-CSF plus IL-4, they differentiated into DCs that exhibited a characteristic irregular shape, formed multiple nonadherent colonies (Fig. 1GoC), and acquired the expression of CD1a in parallel to a progressive loss of expression of CD14 (Table IGo) (42, 48, 49). They were CD83low and CD86low and a minority were CD16+. DCs expressed higher levels of CD80, CD40, HLA-DR, and CD11b, but lower levels of CD11c, compared with M{Phi}s (Table IGo). The combination of GM-CSF plus IL-4 plus IL-10 induced Mo differentiation into adherent cells (DC/IL-10) that exhibited a ramified morphology (Fig. 1GoD). DC/IL-10 expressed similar levels of CD40, CD86, CD11b, and CD11c compared with DCs. The expression of HLA-DR molecules by DC was significantly increased by IL-10: mean fluorescence intensity (MFI) 103.4 ± 35.8 for DCs and 170 ± 51.5 for DC/IL-10 (p = 0.008, Student’s t test). DC/IL-10 expressed low levels of CD1a and did not express CD83 or CD80 (Table IGo). Phenotypic analysis showed that DC/IL-10s are more closely related to DCs than to M{Phi}s, despite their expression of CD16 and CD14 markers (Table IGo). No cellular proliferation was detected in M{Phi}s, M{Phi}/IL-10s, DCs, and DC/IL-10s cultures using a [3H]thymidine uptake assay, and IL-10 did not induce loss of cell viability as determined by trypan blue staining (data not shown).



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  		FIGURE 1. Effect of IL-10 on Mo differentiation into M{Phi}s and DCs. Monocytes were cultured in the presence of M-CSF (M{Phi}) (A), M-CSF plus IL-10 (M{Phi}/IL-10) (B), GM-CSF plus IL-4 (DC) (C), or GM-CSF plus IL-4 plus IL-10 (DC/IL-10) (D) for 6 days. Cell morphology was monitored by light microscopy (x10 magnification). Results are representative of 10 experiments performed with similar results.

 
Replication of primary X4 HIV-1NDK strain in M{Phi}s and DCs

We examined the ability of M{Phi}s and DCs to support productive infection with the primary X4 HIV-1NDK strain. As shown in Fig. 2Go, M{Phi}s infected with HIV-1NDK replicated the virus to high levels, with p24 levels reaching 13 792 pg/ml at day 12 postinfection. In contrast, low levels of viral replication occurred in DC cultures infected with HIV-1NDK, with p24 levels reaching only 40 pg/ml at day 12 postinfection (Fig. 2Go). To test the ability of M{Phi}s and DCs to transmit virus to CD4+ T lymphocytes, HIV-1NDK-infected M{Phi}s and DCs were cocultured with PHA-stimulated autologous CD4+ T cells. The results indicated a significant increase in HIV-1NDK replication in DC:T cell cocultures (1088-fold higher compared with DC alone), with p24 levels reaching 43,520 pg/ml at day 12 postinfection (Fig. 2Go). This increased viral replication was strictly dependent on physical contact between the two cell types, as determined by using a transwell system (data not shown). No significant change in viral production was observed when M{Phi}s were cocultured with CD4+ T cells (13 792 pg/ml vs 14 544 pg/ml, at day 12 postinfection) (Fig. 2Go). However, M{Phi} transmission of virus to PHA-stimulated CD4+ T cells could be detected when they were infected with lower doses of HIV-1NDK (4 ng/ml instead of 20 ng/ml) or with R5 HIV-1 strains (data not shown). These results indicate that despite very low levels of viral replication, DCs transmitted the virus to CD4+ T cells more efficiently than M{Phi}s, which can support high levels of virus replication in the absence of T cell cocultures.



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  		FIGURE 2. Replication of primary X4 HIV-1NDK strain by M{Phi}s and DCs alone or cocultured with autologous CD4+ T cells. M{Phi}s and DCs obtained from the same donor as described in Materials and Methods were infected with HIV-1NDK (20 ng/ml/106 cells) for 12 h, washed, and recultured in the absence or presence of autologous CD4+ T lymphocytes, previously stimulated with PHA (0, 5 µg/ml) plus IL-2 (10 U/ml). The cocultures were performed with a M{Phi}/DC:T ratio of 1:1. Each infection was performed in duplicate. Culture supernatants were collected every 3 days and duplicates were pooled and assessed for p24 Ag. The results are representative of five similar experiments performed.

 
IL-10 inhibits replication of primary X4 HIV-1 strains in M{Phi}

We investigated the effect of IL-10 on viral production by M{Phi}s infected with HIV-1NDK (20 ng/ml/106 cells). IL-10 was present in the cell cultures during the differentiation and infection periods. The results in Fig. 3GoA show that IL-10 inhibited viral replication by M{Phi}s. The p24 levels decreased from 3.49 log in the absence of IL-10 to 0.04 log in the presence of IL-10, at day 12 postinfection. Parallel experiments were performed using another primary X4 HIV-1 strain, VN44 (20 ng/ml/106 cells). IL-10 treatment also inhibited replication of HIV-1VN44 in M{Phi}s (data not shown). We also examined the HIV DNA content in HIV-1NDK-infected M{Phi}s and M{Phi}/IL-10s at 24 h postinfection. We detected 104 copies of Pol HIV-1 DNA/106 cells in M{Phi}s and 102 DNA copies/106 cells in M{Phi}/IL-10s (Fig. 3GoB), indicating that M{Phi}/IL-10s harbored 2 log less HIV DNA copies compared with M{Phi}s. A similar inhibitory effect of IL-10 on HIV DNA copy number was observed when M{Phi}s were infected with HIV-1VN44 (data not shown).



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  		FIGURE 3. Inhibitory effect of IL-10 on HIV-1NDK replication and viral DNA content in M{Phi}s. M{Phi}s were differentiated and infected with HIV-1NDK (20 ng/ml/106 cells) in the presence or absence of IL-10 (10 ng/ml) (see Materials and Methods). Each infection was performed in duplicate. Culture supernatants were collected every 3 days and duplicates were pooled and assessed for p24 Ag. A, Values are the mean ± SD of three experiments performed with cells provided from different donors. M{Phi}s and M{Phi}/IL-10s were infected with HIV-1NDK (20 ng/ml/106 cells) for 12 h. At 24 h postinfection, relative Pol HIV DNA content was assessed by nested PCR in limiting dilution of infected cell lysates in uninfected CEM cell lysates. {beta}-Globin DNA level was assessed and used as a control for DNA content. The PCR sensitivity was 1 copy/3 x 104 cells, as determined in parallel experiments by serial dilutions of 8E5/LAV cells (1 copy HIV DNA/cell) in HIV- CEM cells. B, Results of one representative of three similar experiments performed are presented.

 
IL-10 regulation of CD4 and CXCR4 expression in M{Phi}s

To determine whether IL-10 decreased X4 HIV-1 replication and viral DNA content in M{Phi}s by diminishing their permissiveness to virus entry, we examined the expression of CD4 and CXCR4 molecules on M{Phi}s and M{Phi}/IL-10s. Flow cytometry analysis showed that M{Phi}s expressed both CD4 (82%, MFI 26) and CXCR4 (92%, MFI 53). IL-10 induced up-regulation of both CD4 (96%, MFI 39) and CXCR4 (99%, MFI 128) (Fig. 4Go). The discrepancy between the up-regulation of CD4/CXCR4 expression on M{Phi}/IL-10s and inhibition of viral replication suggests that reduced viral replication in M{Phi}/IL-10s may result from either a postentry block and/or an effect of IL-10 on biochemical properties of CXCR4.



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  		FIGURE 4. Effects of IL-10 on CD4/CXCR4 expression. M{Phi}s and M{Phi}/IL-10s were stained with anti-CD4-PE and anti-CXCR4-PE mAbs. The expression of CD4 (left) and CXCR4 (right) molecules on M{Phi}s and M{Phi}/IL-10s was determined by flow cytometry analysis. Dot plot analyses from 1 experiment representative of 10 independent experiments are shown.

 
To gain insights into the mechanism of CXCR4 regulation by IL-10 in M{Phi}s, we analyzed CXCR4 mRNA and protein expression in M{Phi}s and M{Phi}/IL-10s, by RT-PCR and flow cytometry analysis, respectively. The results indicated an increase in CXCR4 mRNA expression in M{Phi}/IL-10s compared with M{Phi}s (Fig. 5GoA). Treatment of M{Phi}/IL-10s with actinomycin D (AD) (1 µg/ml) diminished CXCR4 mRNA levels (Fig. 5Go A) but induced only a slight decrease in intracellular protein expression (Fig. 5GoB). Cell surface expression of CXCR4 was similar in M{Phi}/IL-10s in the presence or absence of AD (Fig. 5GoB). These results suggest that IL-10 regulates CXCR4 expression in M{Phi}s at the translation or posttranslation level rather than at the transcription level.



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  		FIGURE 5. IL-10 regulation of CXCR4 mRNA and protein expression in M{Phi}s. M{Phi}s and M{Phi}/IL-10s were obtained as described in Materials and Methods. At day 6 of differentiation, M{Phi}/IL-10s were incubated in the presence (M{Phi}/IL-10 plus AD) or absence of AD (1 µg/ml). The level of CXCR4 mRNA and protein expression by M{Phi}/IL-10 was tested after 4 and 8 h of incubation with AD, respectively. Total RNA was extracted from M{Phi}s, M{Phi}/IL-10s, and M{Phi}/IL-10 plus AD, and analyzed by RT-PCR for CXCR4 and GAPDH gene expression. The amplified PCR products of CXCR4 and GAPDH cDNA are shown. A, Results of one representative of three similar experiments performed on cells from different donors are presented. B, M{Phi}s, M{Phi}/IL-10s, and M{Phi}/IL-10 plus AD were stained with anti-CXCR4-PE mAbs (left: surface). An aliquot of these cells was fixed with a 4% formaldehyde buffer and subsequently stained with the same anti-CXCR4 mAbs in the presence of a saponin buffer (right: surface plus intracellular). The expression of CXCR4 molecules was determined by flow cytometry analysis. Dot plot analyses from one experiment representative of five independent experiments performed on cells from different donors are shown.

 
IL-10 increases replication of primary X4 HIV-1 strains in DCs

We then investigated the effect of IL-10 on HIV-1NDK replication by DCs. Viral production was assessed in culture supernatants of DCs cultured and infected in the presence or absence of IL-10 with HIV-1NDK (20 ng/ml/106 cells). Surprisingly, in contrast to the inhibitory effect of IL-10 on HIV-1NDK replication by M{Phi}s, we observed that IL-10 significantly increased viral production by DCs (1.80 log vs 2.99 log, at day 12 postinfection) (Fig. 6GoA). Similar experiments were performed using another primary X4 HIV-1 strain, VN44 (20 ng/ml/106 cells). IL-10 treatment increased HIV-1VN44 replication in DC/IL-10 but decreased viral replication in M{Phi}/IL-10 (data not shown). These results together with the preceding experiments demonstrate that IL-10 has opposite effects on the replication of X4 HIV-1 strains in M{Phi}s and DCs. We next determined the HIV DNA content in HIV-1NDK-infected DCs and DC/IL-10s. We detected 102 copies of Pol HIV DNA/106 cells in DCs and 103 Pol HIV DNA copies/106 cells in DC/IL-10 (Fig. 6GoB), indicating that DC/IL-10 harbored 1 log more HIV DNA copies compared with DCs. A similar effect of IL-10 on viral DNA copy number was observed when DCs were infected with HIV-1VN44 (data not shown). These results indicate that IL-10-mediated stimulatory effects on viral replication are associated with increased viral entry and/or reverse transcription.



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  		FIGURE 6. Stimulatory effect of IL-10 on HIV-1NDK replication and viral DNA content in DCs. DCs were differentiated and infected with HIV-1NDK (20 ngml/106 cells) in the presence or absence of IL-10 (10 ng/ml) (see Materials and Methods). Each infection was performed in duplicate. Culture supernatants were collected every 3 days and duplicates were pooled and assessed for p24 Ag. A, Values are the mean ± SD of three experiments performed with cells from different donors. DCs and DC/IL-10s were then infected with HIV-1NDK (20 ng/ml/106 cells) for 12 h. At 24 h postinfection, relative Pol HIV DNA content was assessed by nested PCR in limiting dilution of infected cell lysates in uninfected CEM cell lysates. B, {beta}-Globin DNA level was assessed and used as a control for DNA content. The PCR sensitivity was 1 copy/3 x 104 cells, as determined in parallel experiments by serial dilutions of 8E5/LAV cells (1 copy HIV DNA/cell) in HIV- CEM cells. Results of one of three similar experiments performed are presented.

 
IL-10 regulation of CD4 and CXCR4 expression in DCs

To determine whether IL-10 increased X4 HIV-1 replication and viral DNA content in DC by increasing their susceptibility to virus entry, we examined the expression of CD4 and CXCR4 molecules in DC cultured in the presence or absence of IL-10. DC expressed high levels of CD4 (95%, MFI 43) but only a low percentage of cells were CXCR4+ (43%, MFI 15) (Fig. 7Go). IL-10 had no effect on CD4 expression (97%, MFI 50) but significantly enhanced the expression of CXCR4 (Fig. 7Go). Thus, the percentage of CXCR4+ cells increased from 38.8 ± 12.2% in the absence of IL-10 (DCs) to 62.9 ± 15.6% in the presence of IL-10 (DC/IL-10) (p < 0,05, Student’s t test).



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  		FIGURE 7. Effects of IL-10 on CD4 and CXCR4 expression by DC. DC and DC/IL-10 were stained with anti-CD4-PE and anti-CXCR4-PE mAbs. The expression of CD4 (left) and CXCR4 (right) on DC and DC/IL-10 was determined by flow cytometry analysis. Dot plot analyses from 1 representative experiment of 10 independent experiments performed are shown.

 
In parallel experiments, we analyzed CXCR4 mRNA and protein expression in DCs and DC/IL-10s. The results indicated an increase of CXCR4 mRNA expression in DC/IL-10s compared with DCs (Fig. 8GoA). Treatment of DC/IL-10s with AD (1 µg/ml) importantly diminished CXCR4 mRNA expression (Fig. 8GoA) and induced a significant decrease in intracellular protein CXCR4 expression (Fig. 8GoB). Cell surface CXCR4 expression was slightly decreased in DC/IL-10s treated with AD compared with DC/IL-10 (Fig. 8GoB). These results suggest that IL-10 regulates CXCR4 expression on DCs at the transcription as well as at the posttranscription level.



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  		FIGURE 8. IL-10 regulation of CXCR4 mRNA and protein expression in DC. DCs and DC/IL-10s were obtained as described in Materials and Methods. At day 6 of differentiation, DC/IL-10 were incubated in the presence (DC/IL-10 plus AD) or absence of AD (1 µg/ml). The level of CXCR4 mRNA and protein expression by DC/IL-10 was tested after 4 and 8 h of incubation with AD, respectively. Total RNA was extracted from DCs, DC/IL-10s, and DC/IL-10 plus AD, and analyzed by RT-PCR for CXCR4 and GAPDH gene expression. The amplified PCR products of CXCR4 and GAPDH cDNA are shown. A, Results of one representative of three similar experiments performed on cells from different donors are presented. B, DCs, DC/IL-10s, and DC/IL-10 plus AD were stained with anti-CXCR4-PE mAbs (left: surface). An aliquot of these cells was fixed with a 4% formaldehyde buffer and subsequently stained with the same anti-CXCR4 mAbs in the presence of a saponin buffer (right: surface plus intracellular). The expression of CXCR4 molecules was determined by flow cytometry analysis. Dot plot analyses from one experiment representative of five independent experiments performed on cells from different donors are shown.

 
Production of RANTES by HIV-1NDK-infected M{Phi}s, M{Phi}/IL-10s, DCs, and DC/IL-10s

We assessed the production of RANTES in culture supernatants of M{Phi}s, M{Phi}/IL-10s, DCs, and DC/IL-10s infected with HIV-1NDK, at day 3 postinfection. We detected in two experiments of three a production of RANTES by HIV-1NDK-infected DCs and more significantly we observed in all experiments a high production of this {beta}-chemokine in DC/IL-10 culture supernatants (Table IIGo). These results suggest that RANTES induces cellular activation via CCR5, thus, favoring X4 HIV-1 strains replication by DC/IL-10. Of note, CCR5 expression is also increased on DCs cultured in the presence of IL-10 (data not shown).


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  		Table II. RANTES production by M{Phi}, M{Phi}/IL-10, DC, and DC/IL-10 infected with HIV-1NDK

 
Effect of IL-10 on HIV-1NDK replication in M{Phi}:T cell and DC:T cell cocultures

We tested the effects of IL-10 on the replication of HIV-1NDK in cocultures of M{Phi}:T cells and DC:T cells. M{Phi}s, M{Phi}/IL-10s, DCs, and DC/IL-10s were infected with HIV-1NDK and then cocultured with PHA-stimulated autologous CD4+ T cells. The results in Fig. 9GoA show that IL-10 efficiently decreased HIV-1NDK replication in M{Phi}:T cell cocultures (3, 3 log vs 1 log at day 12 postinfection). In contrast, IL-10 had no significant effect on HIV-1NDK replication in DC:T cell cocultures (5, 2 log vs 5, 3 log at day 12 postinfection) (Fig. 9GoB). In control experiments, we tested the effect of IL-10 and IL-10 plus IL-4 on viral replication by CD4+ T cells. IL-10 had no significant effect on the level of HIV-1NDK replication by CD4+ T cells compared with lymphocytes cultured in medium alone (4, 8 log vs 4, 9 log at day 8 postinfection). In contrast, the addition of IL-10 plus IL-4 increased viral production by CD4+ T cells compared with IL-10-treated cells (4, 9 vs 5, 3 log at day 9 postinfection) (data not shown). These findings demonstrate that IL-10 decreases replication of X4 HIV-1 strains in M{Phi}:T cell cocultures but has no significant effect on viral replication in DC:T cell cocultures.



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  		FIGURE 9. Effects of IL-10 on HIV-1NDK replication by M{Phi}:T and DC:T cell cocultures. M{Phi}s, M{Phi}/IL-10s (A), DCs, and DC/IL-10s (B) were infected with HIV-1NDK (20 ngml/106 cells), washed, and cocultured with PHA-stimulated autologous CD4+ T cells in the absence or presence of IL-10 (10 ng/ml). The cocultures were performed using a M{Phi}/DC:T cell ratio of 1:1. Each infection was performed in duplicate. Culture supernatants were collected every 3 days and the duplicates were pooled and assessed for p24 Ag. The results are representative of five similar experiments performed on cells from different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A Th2 cytokine pattern has been associated with acquisition of the X4 HIV-1 phenotype in infected patients (36). The effect of Th2 cytokines on the replication of X4 HIV-1 strains by primary cells has not been fully elucidated. IL-10 is known to skew the Th1/Th2 balance to a Th2 profile by selectively blocking IL-12 synthesis in DC (50). The frequency of IL-10-producing CD4+ T cells (51, 52, 53, 54) and IL-10 production by in vitro stimulated PBMC are increased in patients at advanced stages of HIV-1 disease (55). Serum levels of IL-10 also have been reported to be increased in HIV-infected patients with disease progression (37). In vitro experiments demonstrated that HIV-1 infection up-regulates IL-10 expression in Mo/M{Phi} (56). Many reports have demonstrated that IL-10 decreases the replication of M{Phi}-tropic HIV-1 strains in Mo/M{Phi} (35, 43, 56, 57, 58, 59, 60, 61, 62) despite up-regulation of CCR5 expression (32, 63). In contrast, IL-10 at lower concentrations can act in synergy with several cytokines to enhance viral replication by cells of the monocytic lineage (64, 65).

In the present study, we tested the effect of IL-10 on the susceptibility of M{Phi}s and DCs to infection with primary X4 HIV-1 strains and found that IL-10 significantly increased replication of X4 HIV-1 strains in DCs but abolished viral replication in M{Phi}s. To mimic the IL-10 microenvironment present in HIV-infected patients at late stages of disease, we performed experiments in the continuous presence of IL-10 during cell differentiation as well as during the infection and postinfection periods. The morphology and phenotype of M{Phi}/IL-10 was similar to that of M{Phi}s. In contrast, DC/IL-10s were morphologically distinct from DCs. DC/IL-10s were adherent and exhibited a ramified morphology whereas DC exhibited an irregular shape and formed multiple nonadherent colonies. DC/IL-10s also exhibited phenotypic differences compared with DCs including higher levels of HLA-DR, lower levels of CD1a, and lack of CD80 and CD83 expression. DC/IL-10s were CD14+CD16+ compared with DCs, which express low levels of these molecules. Previous studies reported that IL-10 blocks the differentiation of Mos into DCs but promotes their maturation to M{Phi}s, based on the observation that these cells exhibited higher anti-bacterial activity (66) and lower Ag presenting capacities (67). However, we previously demonstrated that the phenotype and the function of DC/IL-10 (CD14+CD162+) remains more closely related to DCs than to M{Phi}s (68). Consistent with this conclusion, Buelens et al. (69) suggested that IL-10 only prevents the maturation of DCs triggered by inflammatory events (e.g., LPS). In contrast, when DC maturation involves CD40-dependent interactions with T cells, the selective inhibition of IL-12 synthesis by IL-10 would favor the development of Th2-type responses (69). Consistent with these findings, we and others suggest that Mos differentiated in the presence of GM-CSF plus IL-4 plus IL-10 exhibit phenotypic and functional DC-like characteristics (68, 70).

When M{Phi}s and DCs were cultured without IL-10, M{Phi}s efficiently replicated X4 HIV-1NDK, whereas low levels of viral replication were detected in infected DCs. The expression of CXCR4 was higher on M{Phi}s than on DCs, suggesting that M{Phi}s may be more permissive for CXCR4-dependent virus entry. These results are in agreement with previous studies that demonstrated the expression of functional CXCR4 HIV coreceptor on M{Phi}s and DCs and the ability of these cells to be productively infected by primary X4 HIV-1 strains (24, 25).

IL-10 significantly increased replication of HIV-1NDK by DCs at a concentration that abolished viral replication by M{Phi}s. HIV DNA copy number directly correlated with changes in HIV p24 levels in both M{Phi}/IL-10 and DC/IL-10, compared with M{Phi}s and DCs, respectively. The inhibition of viral replication in M{Phi}s was not due to an effect of IL-10 on cell viability, because the long-term viability of M{Phi}/IL-10 was slightly increased compared with that of M{Phi}s. We further demonstrated that the opposite effects of IL-10 on viral replication by M{Phi}s and DCs were not restricted to the HIV-1NDK strain, because similar results were obtained with another primary X4 HIV-1 strain, VN44. The opposite effects of IL-10 on viral replication by M{Phi}s and DCs were not due to differential regulation of CD4/CXCR4 expression, because the expression of these molecules was up-regulated by IL-10 in both cell types. Furthermore, IL-10 up-regulated CXCR4 mRNA expression on both cell types.

Treatment of M{Phi}s with AD partially decreased CXCR4 mRNA levels in M{Phi}/IL-10 without inducing a significant decrease in CXCR4 protein expression. This finding may be explained in part by a high stability of CXCR4 mRNA in M{Phi}/IL-10. These results also indicate that in M{Phi}s, IL-10-induced increase of surface CXCR4 expression is not directly correlated with CXCR4 mRNA levels, suggesting a possible effect of IL-10 on CXCR4 protein synthesis and/or membrane turnover. IL-10 has been shown to decrease fluid phase pinocytosis and mannose receptor-mediated uptake in M{Phi}s (71). Thus, one possibility is that IL-10 may enhance surface CXCR4 expression by inducing protein accumulation because of a decreased membrane turnover. Recently, Lapham et al. (72) reported that CXCR4 monomers but not high m.w. CXCR4 species can associate efficiently with CD4 and mediate entry of X4 HIV strains. Taken together, these observations raise the possibility that the inhibitory effect of IL-10 on X4 HIV-1 strain replication by M{Phi}s may be a consequence of an alteration in the biochemical properties of CXCR4 molecules or their ability to associate with CD4. This hypothesis remains to be investigated. In contrast to M{Phi}s, AD treatment of DC/IL-10 nearly abolished CXCR4 mRNA expression in addition to diminishing protein expression, suggesting that IL-10 regulates CXCR4 expression at the transcription level. However, the enhancing effect of IL-10 on CXCR4 expression may not be the only mechanism responsible for increased X4 HIV-1 replication by DC/IL-10. It is possible that IL-10 may have indirect effects on the efficiency of viral entry. Kinter et al. (73) reported that RANTES mediates enhancement of X4 HIV-1 strain replication by increasing the colocalization of CD4 and CXCR4 on primary CD4+ T cells. Therefore, we tested the production of RANTES by X4 HIV-1-infected M{Phi}s, M{Phi}/IL-10, DCs, and DC/IL-10 and found that IL-10 induced high levels of RANTES production in DCs but not in M{Phi}s. Together, these findings raise the possibility that IL-10 may favor CXCR4/CD4 association and entry of X4 HIV-1 strains in DCs as a consequence of cellular activation induced by RANTES via CCR5. It is also possible that IL-10 may have additional effects on early postentry events in the viral life cycle. These hypotheses remain to be investigated. Taken together, our results suggest that viral replication in M{Phi}s and DCs is dependent on distinct cellular factors or pathways differentially regulated by IL-10. Furthermore, using similar culture conditions, we have observed that IL-10 decreased R5 HIV-1 strain replication by both DCs and M{Phi}s. Thus, replication of X4 and R5 HIV-1 strains in DCs is differentially regulated (unpublished observations).

In vivo, both M{Phi}s and DCs interact with T cells in the microenvironment of lymphoid tissue. We demonstrated that DCs but not M{Phi}s exhibit an extraordinary capacity to transmit virus to CD4+ T cells, consistent with previous studies (16, 17, 28, 74, 75). In DC:T cell cocultures, viral production increased 1088-fold compared with that in DCs alone. In contrast, no significant increase was detected in viral production in M{Phi}:T cell cocultures compared with M{Phi}s alone. In the presence of IL-10, we found complete inhibition of viral production in M{Phi}:T cell cocultures. IL-10 did not inhibit HIV-1NDK replication in CD4+ T cells alone, in agreement with Patterson et al. (39). Therefore, the inhibition of HIV-1NDK replication in M{Phi}/IL-10:T cell cocultures by IL-10 is likely to be a consequence of an inhibitory effect on viral replication in M{Phi}s. Of note, IL-10 did not alter the capacity of DCs to transmit virus to CD4+ T cells because viral replication in DC/IL-10:T cell cocultures was similar to that in DC:T cell cocultures. This finding is consistent with our previous study demonstrating that Mos differentiated in the presence of GM-CSF plus IL-4 plus IL-10 exhibit DC-like functional characteristics (68, 70). In contrast to our findings, Weissman et al. (34) demonstrated the capacity of IL-10 to block the replication of the laboratory adapted HIV-1IIIB strain in DC:T cell cocultures. Possible explanations for this discrepancy include differences in cell culture methods used to generate DCs, and differences between primary vs laboratory-adapted X4 HIV-1 strains.

In conclusion, our results demonstrate that IL-10 inhibits the replication of primary X4 HIV-1 strains in M{Phi}s and M{Phi}:T cell cocultures but significantly increases viral replication in DCs without affecting viral production in DC:T cell cocultures. The inhibitory effect of IL-10 on viral replication by M{Phi}s could occur during entry and/or postentry steps. In contrast, IL-10 may enhance the susceptibility of DCs to infection with X4 HIV-1 strains during entry and possibly other early steps in the virus life cycle. To our knowledge, this is the first demonstration that IL-10 differentially regulates X4 HIV-1 replication by M{Phi}s and DCs. The molecular mechanisms for this differential effect remain to be determined. Our studies suggest that IL-10 may stimulate the ability of DCs to propagate X4 HIV-1 strains in vivo and support the hypothesis that IL-10 may favor the replication of X4-HIV-1 strains as HIV-infected individuals progress to AIDS.


   Acknowledgments
 
We thank Dr. Françoise Barré-Sinoussi (Pasteur Institute, Paris, France) for providing us X4 HIV-1NDK and HIV-1VN44 strains. We also thank Dr. Michel Goldman (Faculty of Medicine, Brussels, Belgium) for critical review of the manuscript and Michel Paing (Broussais Hospital, Paris, France) for editorial assistance.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale, SIDACTION, and Agence Nationale de la Recherche sur le SIDA. P.A. is a recipient of a grant from SIDACTION, Y.B. and H.H. from Agence Nationale de la Recherche sur le SIDA, and N.C. from the French Research and Education Ministry. D.G. is an Elizabeth Glaser Scientist supported by the Pediatric AIDS Foundation. Back

2 Address correspondence and reprint requests to Dr. Petronela Ancuta, Unité d’Immunopathologie Humaine, Institut National de la Santé et de la Recherche Médicale U430, Hôpital Broussais, 96 rue Didot, 75674, Paris, Cedex 14, France. Back

3 Abbreviations used in this paper: M{Phi}, macrophage; CXCR, CXC chemokine receptor; X4 HIV-1, CXCR4-dependent HIV-1; DC, dendritic cell; Mo, monocyte; R5 HIV-1, CCR5-dependent HIV-1; AD, actinomycin D; MFI, mean fluorescence intensity. Back

Received for publication July 27, 2000. Accepted for publication January 16, 2001.


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