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The Journal of Immunology, 2000, 164: 1582-1587.
Copyright © 2000 by The American Association of Immunologists

Retrovirally Mediated IFN-ß Transduction of Macrophages Induces Resistance to HIV, Correlated with Up-Regulation of RANTES Production and Down-Regulation of C-C Chemokine Receptor-5 Expression1

Isabelle Cremer2, Vincent Vieillard3 and Edward De Maeyer

Equipe de l’Interferon et des Cytokines, Unité Mixte de Recherche 146, Centre National de la Recherche Scientifique Institut Curie, Orsay, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Constitutive expression of IFN-ß by HIV target cells may be an alternative or complementary therapeutic approach for the treatment of AIDS. We show that macrophages derived from CD34+ cells from umbilical cord blood can be efficiently transduced by a retroviral vector carrying the IFN-ß coding sequence. This results in resistance to infection by a macrophage-tropic HIV type 1, as shown by the drastic reduction in the HIV DNA copy number per cell and in p24 release. Moreover, IFN-ß transduction totally blocked secretion of proinflammatory cytokines after HIV infection. The constitutive IFN-ß production also resulted in an increased production of IL-12 and IFN-{gamma} Th1-type cytokines and of the ß-chemokines macrophage-inflammatory protein-1{alpha}, macrophage-inflammatory protein-1ß, and RANTES. RANTES was found to be involved in the HIV resistance observed, and this was correlated with a down-regulation of the CCR-5 HIV entry coreceptor. These results demonstrate the feasibility and the efficacy of such IFN-ß-mediated gene therapy. In addition to inhibiting HIV replication, IFN-ß transduction could have beneficial immune effects in HIV-infected patients by favoring cellular immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocytes and macrophages are key players in the pathogenesis of HIV-1 infection (1, 2). They are among the first cells to be infected by HIV-1. Unlike lymphocytes, HIV-infected-macrophages do not die, they persist in tissues for long periods, and they are capable of producing large amounts of HIV. Thus, they are major reservoirs for HIV during all stages of the disease and represent an efficient vector for viral dissemination throughout the body (3). The replication of HIV in tissue macrophages has been associated with clinical manifestations, including encephalopathy (1). Macrophages are also targets for opportunistic infections such as herpes virus type 1 or Mycobacterium tuberculosis during the course of HIV disease (4). Moreover, HIV-infected macrophages show impaired antimicrobial activity and increased production of the proinflammatory cytokines IL-1, TNF-{alpha}, and IL-6 (2, 5), which are potent up-regulators of HIV replication. Thus, HIV infection of monocytes and macrophages plays a critical role in the pathogenesis of AIDS.

The eradication of HIV from infected persons is the ultimate goal of HIV therapeutic interventions. Progress has been made in developing antiretroviral molecules that suppress HIV replication, and tritherapy treatment was almost successful in that viral load is not detectable in treated individuals (6). However, during the treatment, a low replication of HIV goes on in lymphoid organs. In the present work, our design consisted of producing an anti-HIV resistant state in macrophages as a therapeutic approach to HIV disease through a low continuous production of IFN-ß that affects several stages of the HIV life cycle in infected macrophages (7, 8, 9, 10) and results in inhibition of HIV replication. For this purpose, macrophages were transduced by a retroviral vector (HMB-KbHuIFNß) carrying the human IFN-ß coding sequence driven by a fragment of the H-2Kb MHC gene promoter (11). Gene modification of macrophages has been achieved by transducing highly proliferating progenitor cells. Purified CD34+ cells from umbilical cord blood were first amplified in the presence of IL-3, IL-6, and stem cell factor (SCF)4 were retrovirally transduced by coculture with irradiated producer lines, and were then differentiated into macrophages.

We show that IFN-ß transduction of macrophages induces anti-HIV-YU-2 resistance, which is correlated with an increased RANTES production and a down-regulation of CCR-5 chemokine receptor expression. IFN-ß transduction of macrophages also induced an increased production of the Th1-type cytokines IL-12 and IFN-{gamma} and of the ß-chemokines macrophage inflammatory protein (MIP)-1{alpha} and MIP-1ß. Moreover, no proinflammatory cytokine production (IL-1{alpha} and TNF-{alpha}) was detected in HIV-infected macrophages after IFN-ß transduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection and purification of cord blood CD34+ cells

Umbilical cord blood samples were obtained from consenting mothers at the maternity ward of the Orsay Hospital. Mononuclear cells were isolated by Ficoll-Paque Plus (Pharmacia Biotech, Orsay, France) density gradient centrifugation and cells bearing CD34 Ag were isolated using the CD34 isolation kit (QBEND/10; Minimacs separation columns, Miltenyi Biotec, Bergisch Gladbach, Germany). After purification, CD34+ cells were prestimulated with cytokines IL-3 (200 U/ml; R&D Systems, Abingdon, U.K.), IL-6 (500 U/ml; PeproTech, London, U.K.), and SCF (40 U/ml; R&D Systems) in IMDM (Life Technologies, Cergy Pontoise, France) supplemented with 10% of heat-inactivated FCS (HyClone, Erembodegem Aalst, Belgium). Flow cytometric analysis demonstrated a purity of >99% CD34+ cells.

Retroviral transduction and differentiation of macrophages from CD34+

{Psi}-CRIP-HMB-KbHuIFNß packaging cells used for IFN-ß gene transduction were obtained as previously described (11). Briefly, the pHMB-KbHuIFNß vector (11) was introduced into cells of the {Psi}-CRIP fibroblastic line (12) by electroporation. The packaging clone selected produced 105 retroviral particles/ml. The absence of helper virus production by packaging clones was confirmed by using a sensitive marker rescue assay based on a LacZ reporter gene. After a culture period of 15 days in IMDM containing IL-3, IL-6, and SCF for expansion, 5 x 105 CD34+-derived proliferating cells were transduced in a 2-day coculture on irradiated (5000 rad) {Psi}-CRIP-HMB-KbHuIFNß packaging cells (11) that were grown to 50% confluence in IMDM supplemented with 5% FCS, 5% newborn calf serum (HyClone), 10 µg/ml protamine sulfate (Sigma-Aldrich, St. Quentin Fallavier, France), IL-3, IL-6, and SCF. Control cells were cocultured on {Psi}-CRIP or {Psi}-CRIP-HMB-neo packaging cells, producing the retroviral vector coding for the neomycin phosphotransferase gene (13). Nonadherent cells were then removed from packaging cells and cultured for additional 3 days in IMDM medium supplemented with 10% FCS, IL-3, IL-6, and SCF. Macrophages were generated in RPMI 1640 medium (Life Technologies) in the presence of GM-CSF (100 ng/ml; Schering-Plough, Levallois-Perret, France) and 10% human serum AB (Centre National de la Transfusion Sanguine, Rungis, France) over 2 or 3 wk.

Cytofluorometric cell surface phenotyping

Macrophage-like cells were processed for single staining using FITC- or PE- conjugated mAbs. The cells were incubated for 20 min in PBS buffer containing 20% serum AB and were stained for 1 h with the following conjugated Abs: FITC-labeled anti-CD4 (Becton Dickinson, Le-Pont-de-Claix, France), anti-HLA-DR (PharMingen, Le-Pont-de-Claix, France), anti-HLA-ABC (Coulter, Margency, France), anti-CCR-5, anti-CXCR-4 (R&D Systems), or PE-labeled anti-CD14 (Becton Dickinson). Negative controls were performed with mismatched mAbs (Becton Dickinson). Fluorescence analysis was determined with a FACScan flow-cytometer and CellQuest software (Becton Dickinson).

HIV resistance analysis

HIV-YU-2 virus stock was prepared as previously described (14). Briefly, COS-1 cells were transfected with the plasmid containing the HIV-YU-2 DNA sequence (15) and were cocultured with PBL for 6 days. Infected PBL were removed from COS cells, and fresh uninfected PBL were added every 3 days. The cell supernatant from infected PBL was collected 15 days later and stored at -80°C. This HIV-YU-2 stock contained 40 ng/ml p24 and an infectious titer of 2.5 x 105/ml TCID50. Untransduced, neo-transduced, or IFN-ß-transduced macrophages were seeded in 6-well plates at a concentration of 5 x 105 cells/ml and HIV-YU-2 was added for 3 h at 37°C and at a multiplicity of infection close to 0.01 in the presence of 10 µg/ml of protamine sulfate. The cells were washed two times in PBS, and fresh medium was added. Uninfected cell populations were run in parallel. We determined IFN production using a biological assay (14), cytokine production by ELISA (R&D Systems), the proportion of HIV DNA copies by PCR amplification, and virus released into the culture supernatants by ELISA for HIV p24 Ag at different times after infection (Dupont de Nemours, Les Ulis, France).

PCR analysis for detection of IFN-ß transgene integrations and HIV DNA copies

The numbers of HIV DNA copies and IFN-ß transgene integrations were estimated as previously described (14). The relative intensity of the bands was compared with the serial 2-fold dilutions of the reference bands obtained with the DNA preparations derived from plasmid-transfected U937 cells containing one copy of IFN-ß transgene per cell (16) or J. Jhan cells containing one copy of HIV DNA (17). The absence of murine packaging cells was verified by PCR analysis with a murin {alpha}-globin set primer (18).

Quantification of cytokines by ELISA

The cytokines and chemokines IL-1{alpha}, TNF-{alpha}, IFN-{gamma}, IL-12, MIP-1{alpha}, MIP-1ß, and RANTES were quantified from cell-free supernatants of macrophages using ELISA kits (Quantikine) purchased from R&D Systems.

RT-PCR analysis of chemokine receptor expression

Total RNAs were isolated from macrophages as previously described (19). cDNA products were obtained from 1 µg of total RNA using the First Strand Synthesis kit (Pharmacia Biotech). One-sixteenth of the cDNA products were amplified by PCR for 35 cycles in the presence of 1 µM [33P]{alpha} dCTP to detect the human glyceraldehyde-3-phosphate dehydrogenase transcripts as a quantitative control. To estimate chemokine receptor expression, cDNA products were amplified for 40 cycles using the following primers: a CXCR-4 primer set 5'-ACGTCAGTGAGGCAGATG-3' sense and 5'- GATGACTGTGGTCTTGAG-3' antisense and a CCR-5 primer set 5'-GTCCAATCTATGACATCA-3' sense and 5'-GGTGTAATGAAGACCTTC-3' antisense. The reaction products were detected by autoradiography after electrophoresis on 4% nondenaturing polyacrylamide gels and were quantified using the PhosphorImager (Molecular Dynamics Sevenoaks, U.K.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retrovirally mediated IFN-ß transduction of macrophages confers high anti-HIVYU-2 resistance

To obtain high numbers of IFN-ß-transduced macrophages, CD34+ cells isolated from umbilical cord blood were cultured for 2 wk in the presence of IL-3, IL-6, and SCF. Highly proliferating cells were then transduced with HMB-KbHuIFN-ß or HMB-neo retroviral vectors and differentiated into macrophages by culturing them with GM-CSF and human serum. We reproducibly obtained high average transduction efficiencies, ranging from 50 to 100% as determined by PCR analysis (Table IGo). Because they are averages, these percentages do not imply that one cell of two or that all the cells had been transduced but may mean that <50 or 100% of the cells were transduced with some cells bearing multiple copies of the transgene. Thirteen days after retroviral transduction, IFN-ß-transduced macrophages secreted 480-1045 U/106 cells per 3 days of IFN-ß, whereas untransduced and neo-transduced cells produced no detectable levels of IFN-ß (Table IGo and data not shown). Up to 3 wk after gene transduction, the survival of IFN-ß-producing macrophages was similar to that of untransduced or neo-transduced cells, as determined by trypan blue exclusion test (data not shown). Immunofluorescence analyses revealed that surface Ags expressed by macrophages included CD4, CD14, HLA-ABC, and HLA-DR (Table IGo). Expression of CD4 and CD14 was not modified by IFN-ß transduction, whereas expression of MHC class I and class II Ags was slightly increased (Table IGo). Moreover, both untransduced and IFN-ß-transduced macrophages were able to phagocyte latex beads with similar efficiency (data not shown). We showed that neither retroviral transduction nor low constitutive expression of IFN-ß had any apparent effect on cell differentiation.


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  		Table I. Phenotyping of untransduced (UT) and IFN-ß-transduced (IFN-T) macrophages1

 
To assess the in vitro efficacy of low constitutive expression of IFN-ß on HIV infection, IFN-ß-transduced macrophages were tested for resistance to M-cell-tropic HIV-YU-2 challenge. We observed that untransduced and neo-transduced cells could be productively infected by HIV-YU-2. As shown in Fig. 1Go, p24 Ag secretion in untransduced macrophages rapidly reached high levels (up to 100 ng/ml) since day 3 after infection and was maintained for >15–18 days. In neo-transduced control macrophages, p24 Ag secretion was slightly lower than that of untransduced cells and increased progressively to reach values ranging from 50 to 90 ng/ml (Fig. 1Go, C and D). In contrast, p24 Ag secretion by IFN-ß-transduced macrophages remained extremely low throughout the culture in the 4 donors tested (Fig. 1Go). Furthermore, the infectivity of HIV particles released by IFN-ß-transduced macrophages 9 days after infection (determined on P4-2 Hela cells (20)) was reduced 6-fold compared with that of neo-transduced cells (data not shown). Similar protection against HIV-YU-2 was obtained after addition of 1000 U/ml of recombinant IFN-ß (Fig. 1GoD), further confirming the specific ability of IFN-ß to confer HIV resistance to macrophages. These results were correlated with a drastic reduction of HIV DNA copy number per cell in IFN-ß-transduced macrophages compared with untransduced macrophages. As shown in Fig. 2Go, the number of HIV DNA copy per cell increased from 0.001 at day 3 to 0.5 at day 12 after HIV infection. On the contrary, IFN-ß-transduced macrophages contained a very low HIV DNA copy number per cell (<0.05 at day 12). A similar resistance against the M-tropic HIV-BAL strain was observed. Nine days after HIV-BAL infection, the p24 production in IFN-ß-transduced cells remained at the low level of 5 ng/ml, whereas a p24 production of ~130 ng/ml was detected in untransduced macrophages (data not shown). These results demonstrate that IFN-ß transduction strongly inhibits HIV-YU-2 and HIV-BAL infection of macrophages.



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  		FIGURE 1. Inhibition of HIV-YU-2 replication by IFN-ß transduction of macrophages. A–D represent the cells from four independent donors. About 1 month after IFN-ß transduction, macrophages were infected with HIV. Cell culture supernatants from untransduced (UT), neo-transduced (neo-T), or IFN-ß-transduced (IFN-T) cells or from cells treated with 1000 U/ml of recombinant IFN-ß (rec IFN) were collected at time points indicated, and HIV p24 ELISA was performed.

 


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  		FIGURE 2. IFN-ß transduction of macrophages significantly reduces the number of HIV-YU-2 DNA copies per cell. A and B represent the cells from two independent donors. About 1 month after IFN-ß transduction, macrophages were infected with HIV. DNA was extracted from untransduced (UT) and IFN-ß-transduced (IFN-T) cells at time points indicated, and the number of HIV DNA copies per cell was determined by PCR analysis.

 
IFN-ß transduction enhances Th1-type cytokine and ß-chemokine production by macrophages

Previous reports have demonstrated that type I IFN modulate the production of several immunomodulatory cytokines (7, 11). The secretion of Th1-type and proinflammatory cytokines and of ß-chemokines was thus determined in neo-transduced and IFN-ß-transduced macrophages 9 days after the onset of HIV infection. A similar amount (18 pg/106 cells) of IL-12 was detected in supernatants from HIV-infected and uninfected macrophages (Fig. 3Go), whereas there is a 10-fold increase of IFN-{gamma} production by macrophages after HIV infection. Moreover, in uninfected macrophages, the Th1-type cytokine production was enhanced after IFN-ß transduction. The IL-12 and IFN-{gamma} production were 3-fold and 14-fold higher, respectively, in IFN-ß-transduced compared with neo-transduced (Fig. 3Go) or untransduced macrophages (data not shown). In IFN-ß-transduced macrophages, similar levels of IFN-{gamma} were detected in uninfected and HIV-infected cells. The production of TNF-{alpha} and IL-1{alpha} proinflammatory cytokines was also analyzed in neo-transduced and IFN-ß-transduced cells. TNF-{alpha} production was 23-fold higher after HIV infection, and IL-1{alpha} production, which was undetectable in uninfected cells (<1.5 pg/106 cells), went up to 445 pg/106 cells in HIV-infected cells. On the contrary, IFN-ß transduction of the cells did not modify the production of these proinflammatory cytokines in uninfected or HIV-infected cells, confirming that in these cells HIV replication was inhibited.



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  		FIGURE 3. IFN-ß transduction of macrophages increases the production of Th1-type cytokines and ß-chemokines but not that of proinflammatory cytokines. IFN-{gamma} and IL-12 Th1-type cytokines, IL-1{alpha} and TNF-{alpha} proinflammatory cytokines, and MIP-1{alpha}, MIP-1ß, and RANTES ß-chemokines were quantified by ELISA in the culture medium of neo-transduced (neo-T, gray histograms) or IFN-ß-transduced macrophages (IFN-T, black histograms) in uninfected (UI) and HIV-infected cells (HIV). These results are representative of three independent experiments.

 
Several reports indicate that HIV infection of macrophages results in an increased production of the ß-chemokines which are ligands for the chemokine receptor CCR-5 (21, 22). Therefore, we compared RANTES, MIP-1{alpha}, and MIP-1ß production in neo-transduced and IFN-ß-transduced macrophages after HIV infection. The production of MIP-1{alpha} and MIP-1ß was increased 4-fold and 6-fold, respectively, by HIV infection (Fig. 3Go). IFN-ß transduction induced an enhanced production of these ß-chemokines (6-fold, 2-fold, and 4-fold for RANTES, MIP-1{alpha}, and MIP-1ß, respectively) that was not modified after HIV infection of the cells, further confirming that the cells were resistant to HIV-YU-2 infection.

HIV resistance observed in IFN-ß-transduced macrophages could be mediated by RANTES

RANTES is implicated in HIV resistance in several cell types, essentially through a competitive binding and down-regulation of CCR-5, which is the major entry coreceptor for M-cell-tropic strains of HIV. Therefore, we analyzed whether the HIV resistance observed in IFN-ß-transduced macrophages could be mediated by RANTES because we have observed a significant increase of RANTES production after IFN-ß transduction of the cells. As shown in Fig. 4Go, the addition of RANTES to neo-transduced macrophages resulted in HIV resistance. Moreover, the addition of blocking Ab to RANTES in IFN-ß-transduced cell cultures abolished the HIV-YU-2 resistance, indicating that RANTES is required for HIV resistance of IFN-ß-transduced macrophages (Fig. 4Go). We then investigated the ability of IFN-ß to modify the level of chemokine receptor expression. Thus, by RT-PCR and FACS analysis we analyzed the expression of CXCR-4 and CCR-5. As shown in Figs. 5Go and 6, untransduced macrophages expressed both the CXCR-4 and the CCR-5 HIV entry coreceptors. The level of CXCR-4 expression was not modified after HIV infection or after IFN-ß transduction of the cells, whereas we observed a 6-fold reduction of transcripts for CCR-5 in IFN-ß-transduced macrophages compared with untransduced macrophages (Fig. 5Go). Flow cytometry analysis confirmed that a treatment of macrophages with 1000 U/ml of IFN-ß decreased cell surface expression of CCR-5, whereas it had no effect on CXCR-4 expression (Fig. 6Go). These results indicate that IFN-ß-mediated HIV-YU-2 resistance in macrophages may be due to an increased expression of RANTES correlated with a down-regulation of the CCR-5 chemokine receptor.



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  		FIGURE 4. IFN-ß transduction of macrophages significantly reduces the number of HIV-YU-2 DNA copies per cell through RANTES production. Neo-transduced (neo-T) and IFN-ß-transduced (IFN-T) macrophages were infected with HIV-YU-2, and the number of HIV DNA copies per cell was determined 9 days later by PCR analysis. When indicated, macrophages were treated with 10 µg/ml of anti-RANTES mAb (aRANTES) or with 10 ng/ml of recombinant RANTES.

 


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  		FIGURE 5. IFN-ß transduction of macrophages significantly reduces CCR-5 expression. Nine days after HIV infection, RNA was extracted from neo-transduced (neo-T) and IFN-ß-transduced (IFN-T) macrophages in uninfected (UI) and HIV-infected cells (HIV). Detection of CXCR-4 and CCR-5 transcipts was performed by RT-PCR analysis.

 


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  		FIGURE 6. IFN-ß treatment of macrophages reduces cell surface expression of CCR-5. Macrophages were treated with 1000 U/ml of recombinant IFN-ß for 3 days. The levels of CCR-5 and CXCR-4 expression were determined by FACS analysis. Solid lines represent untreated macrophages, dotted lines represent IFN-ß-treated macrophages, and bold lines represent isotype control background staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Murine retroviral vectors are unable to transduce nondividing cells (23), which include terminally differentiated macrophages, one of the major target cells for HIV. One possible means of overcoming this limitation is to access hematopoietic progenitor cells, which give rise to cells of the monocyte/macrophage lineage. The efficient transduction of hematopoietic stem cells derived from HIV-infected patients using an IFN-ß-carrying retroviral vector will require an extensive preparatory investigation. As one approach to this aim, we stimulated CD34+ cells for 15 days with a combination of cytokines before IFN-ß transduction. After this period, the cells were differentiated into macrophages. Using such a protocol, we reproducibly obtained high transduction efficiency (50–100%). In addition, no significant effect was observed on macrophage differentiation or expression of CD4 and CD14 cell surface Ags.

The ability of low constitutive expression of IFN-ß to inhibit viral replication in macrophages was then examined. Our data showed that IFN-ß is effective in inhibiting HIV-YU-2 replication in macrophages, as seen in the observed ~10-fold reduction in viral replication and 100-fold reduction in the number of HIV DNA copy per cell compared with control untransduced or neotransduced cells. This is consistent with our previous observations that constitutive IFN-ß production confers resistance against T-tropic (HIV-BRU) and M-tropic (HIV-YU-2, HIV-BAL) in several cell types, including PBL from HIV-infected donors (11) and CD34+ TF-1 cells (14). Several studies have reported the antiviral effects of type I IFN on macrophages that take place at early and late stages of the HIV infectious cycle (10, 24). The possibility of transducing CD34+-derived macrophages using retroviral vectors encoding for proteins that would interfere with HIV replication has been described. Macrophages expressing a ribozyme gene, a Tat responsive element decoy linked to an antisense tat molecule, or a transdominant mutant HIV-1 RevM10 protein resisted HIV infection in vitro (25, 26, 27).

In HIV-infected control macrophages, an increased secretion of the proinflammatory cytokines TNF-{alpha} and IL-1{alpha} was oberved, contrasting with HIV-infected IFN-ß-transduced macrophages in which the levels of IL-1{alpha} and TNF-{alpha} remained undetectable. Similar observations have been made in IFN-ß-transduced PBL from HIV-infected patients (11). Moreover, elevated levels of proinflammatory cytokines were detected in the serum of HIV-infected patients (28, 29). Because it has been shown that HIV-1 tat protein induces TNF-{alpha}, IL-1{alpha}, and IFN-{gamma} production (30, 31, 32), it is likely that the undetectable level of IL-1{alpha} and TNF-{alpha} in IFN-ß-transduced macrophages reflects resistance to HIV infection.

High levels of production of proinflammatory cytokines are detrimental in the context of AIDS because they can alter immune reponses, cause tissue damage, and up-regulate HIV replication (33). IL-1 and TNF-{alpha} are also involved in the pathogenic mechanisms of Kaposi sarcoma (34, 35, 36) and neurologic disease. Persidsky et al. (37) suggested that the up-regulation of TNF-{alpha}, IL-6, and IL-10 is a major event that permits the transendothelial migration of monocytes into brain tissue, thus expanding the viral reservoir in the brain (38) leading to progressive neurologic impairment that appears at late stages of AIDS. Thus, IFN-ß transduction of macrophages may also be a therapeutic opportunity for the prevention of AIDS-associated dementia because the levels of proinflammatory cytokines in IFN-ß-transduced macrophages are not up-regulated after HIV infection.

We demonstrated that IFN-ß-transduced macrophages secreted 3- and 14-fold more IL-12 and IFN-{gamma}, respectively, compared with untransduced cells. Increased production of Th1-type cytokines was observed after IFN-ß transduction of PBL (11) and dendritic cells.5 Type I IFN are known to increase the frequency of Th1 cells (39, 40, 41, 42). During the progression of AIDS, there is a decreased expression of Th1-type cytokines concomitant with an increased expression of Th2-type cytokines, resulting in altered immune responses (43, 44). Our results show that IFN-ß transduction of macrophages can favor the development of a Th1-type immune response that would be beneficial in HIV-infected patients because it restores Th1-type immune responses.

Concomitant with the increased production of Th1-type cytokines, we also observed that IFN-ß transduction of macrophages enhanced the secretion of the ß-chemokines RANTES, MIP-1{alpha}, and MIP-1ß. HIV infection of macrophages induces an up-regulation of ß-chemokine production, which has been reported by others (21, 22). The enhanced release of ß-chemokines in the tissues by HIV-infected macrophages and by IFN-ß transduction might attract uninfected T cells and monocytes to the site of active infection.

Of the three ß-chemokines capable of inhibiting HIV entry in macrophages (45, 46) through the CCR-5 coreceptor, RANTES is the most efficient. To assess whether RANTES is sufficient to inhibit HIV replication, recombinant RANTES was added before HIV infection of macrophages. As shown in Fig. 5Go, the addition of RANTES inhibited HIV replication as evidenced by the fact that no p24 production was detected. We next investigated whether the RANTES chemokine released in IFN-ß-transduced macrophages played a role in the inhibition of HIV replication. The addition of RANTES-blocking Ab neutralized the inhibitory activity of IFN-ß transduction of macrophages. Thus, our data suggest that the IFN-ß-dependent release of RANTES by macrophages plays major role in the inhibition of HIV replication. The simultaneous neutralization of RANTES, MIP-1{alpha}, and MIP-1ß has been shown to be required to abrogate the HIV-suppressive effects of CD8+ T cells supernatants (46). In previous experiments, we have observed that IFN-ß transduction of CD34+ TF-1 cells results in a protection against HIV-YU-2 infection that is correlated with a 5-fold decrease in CCR-5 expression (14). CCR-5 expression was examined in macrophages and revealed a 6-fold decrease of the mRNA transcripts for CCR-5 in IFN-ß-transduced cells compared with untransduced cells. FACS analysis revealed a decreased expression of CCR-5 after a treatment of macrophages with recombinant IFN-ß. A down-regulation of CCR-5 expression on macrophages in response to IL-4 and IL-13 cytokines has also been reported and was correlated with an inhibition of HIV entry and replication (47). More recently, Lane et al. (48) have shown that TNF-{alpha} inhibits HIV replication in macrophages by inducing the production of RANTES and by decreasing CCR-5 expression. These data suggest that several cytokines are strongly implicated to prevent HIV infection of macrophages by increasing RANTES production and by decreasing CCR-5 expression. Thus, the resistance we have observed against HIV infection is most likely a consequence of the multiple antiretroviral activities resulting from IFN-ß transduction of macrophages. Our data indicate that low constitutive production of IFN-ß can be used as an approach to somatic-cell gene therapy of HIV infection, to inhibit viral replication, and to improve immune functions.


   Acknowledgments
 
We thank Catherine Sautès-Fridman for critical reading of the manuscript and the personnel of the maternity ward of the Orsay Hospital for providing umbilical cord blood.


   Footnotes
 
1 This work was supported by the Agence Nationale de Recherches sur le SIDA (ANRS) and by the Fondation pour la Recherche Médicale (SIDACTION). I.C. was supported by a fellowship from ANRS. Back

2 Address correspondance and reprint requests to Dr. Isabelle Cremer at her current address: Laboratoire d’Immunologie Cellulaire et Clinique, INSERM U255, Institut Curie, 26 rue d’Ulm, 75005 Paris, France. E-mail address: Back

3 Current address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138. Back

4 Abbreviations used in this paper: SCF, stem cell factor; MIP, macrophage inflammatory protein. Back

5 Cremer, I., V. Vieillard, C. Sautès-Fridman, and E. De Maeyer. Inhibition of HIV transmission to CD4+ T cells after gene transfer of constitutively expressed IFN-ß to dendritic cells. Submitted for publication. Back

Received for publication June 25, 1999. Accepted for publication November 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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