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This information is current as Chemokine Receptor-5 Expression
Production and Down-Regulation of C-C
Correlated with Up-Regulation of RANTES
Macrophages Induces Resistance to HIV,
Transduction ofβRetrovirally Mediated IFN-
Isabelle Cremer, Vincent Vieillard and Edward De Maeyer
http://www.jimmunol.org/content/164/3/1582
doi: 10.4049/jimmunol.164.3.1582
2000; 164:1582-1587; ;J Immunol
References http://www.jimmunol.org/content/164/3/1582.full#ref-list-1
, 32 of which you can access for free at: cites 47 articlesThis article
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2000 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
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Retrovirally Mediated IFN-

Transduction of Macrophages
Induces Resistance to HIV, Correlated with Up-Regulation of
RANTES Production and Down-Regulation of C-C Chemokine
Receptor-5 Expression
1
Isabelle Cremer,
2
Vincent Vieillard,
3
and Edward De Maeyer
Constitutive expression of IFN-

by HIV target cells may be an alternative or complementary therapeutic approach for the
treatment of AIDS. We show that macrophages derived from CD34
ⴙ
cells from umbilical cord blood can be efficiently transduced
by a retroviral vector carrying the IFN-

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-

transduction
totally blocked secretion of proinflammatory cytokines after HIV infection. The constitutive IFN-

production also resulted in an
increased production of IL-12 and IFN-
␥
Th1-type cytokines and of the

-chemokines macrophage-inflammatory protein-1
␣
,
macrophage-inflammatory protein-1

, 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-

-mediated gene therapy. In addition to inhibiting HIV replication, IFN-

transduction could have beneficial
immune effects in HIV-infected patients by favoring cellular immune responses. The Journal of Immunology, 2000, 164: 1582–
1587.
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 manifesta-
tions, including encephalopathy (1). Macrophages are also targets
for opportunistic infections such as herpes virus type 1 or Myco-
bacterium tuberculosis during the course of HIV disease (4).
Moreover, HIV-infected macrophages show impaired antimicro-
bial activity and increased production of the proinflammatory cy-
tokines IL-1, TNF-
␣
, and IL-6 (2, 5), which are potent up-regu-
lators 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 treat-
ment, a low replication of HIV goes on in lymphoid organs. In the
present work, our design consisted of producing an anti-HIV re-
sistant state in macrophages as a therapeutic approach to HIV dis-
ease through a low continuous production of IFN-

that affects
several stages of the HIV life cycle in infected macrophages (7–
10) and results in inhibition of HIV replication. For this purpose,
macrophages were transduced by a retroviral vector (HMB-
K
b
HuIFN

) carrying the human IFN-

coding sequence driven by
a fragment of the H-2K
b
MHC gene promoter (11). Gene modifi-
cation 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 irradi-
ated producer lines, and were then differentiated into macrophages.
We show that IFN-

transduction of macrophages induces anti-
HIV-YU-2 resistance, which is correlated with an increased RAN-
TES production and a down-regulation of CCR-5 chemokine re-
ceptor expression. IFN-

transduction of macrophages also
induced an increased production of the Th1-type cytokines IL-12
and IFN-
␥
and of the

-chemokines macrophage inflammatory
protein (MIP)-1
␣
and MIP-1

. Moreover, no proinflammatory cy-
tokine production (IL-1
␣
and TNF-
␣
) was detected in HIV-in-
fected macrophages after IFN-

transduction.
Materials and Methods
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,
Equipe de l’Interferon et des Cytokines, Unite´ Mixte de Recherche 146, Centre Na-
tional de la Recherche Scientifique Institut Curie, Orsay, France
Received for publication June 25, 1999. Accepted for publication November
15, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Agence Nationale de Recherches sur le SIDA
(ANRS) and by the Fondation pour la Recherche Me´dicale (SIDACTION). I.C. was
supported by a fellowship from ANRS.
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: Isabelle.Cremer@curie.fr
3
Current address: Department of Molecular and Cellular Biology, Harvard Univer-
sity, Cambridge, MA 02138.
4
Abbreviations used in this paper: SCF, stem cell factor; MIP, macrophage inflam-
matory protein.
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00
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Bergisch Gladbach, Germany). After purification, CD34
⫹
cells were pre-
stimulated 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) sup-
plemented 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
⫹
⌿-CRIP-HMB-K
b
HuIFN

packaging cells used for IFN-

gene transduc-
tion were obtained as previously described (11). Briefly, the pHMB-
K
b
HuIFN

vector (11) was introduced into cells of the ⌿-CRIP fibroblas-
tic line (12) by electroporation. The packaging clone selected produced 10
5
retroviral particles/ml. The absence of helper virus production by packag-
ing clones was confirmed by using a sensitive marker rescue assay based
onaLacZ reporter gene. After a culture period of 15 days in IMDM
containing IL-3, IL-6, and SCF for expansion, 5 ⫻10
5
CD34
⫹
-derived
proliferating cells were transduced in a 2-day coculture on irradiated (5000
rad) ⌿-CRIP-HMB-K
b
HuIFN

packaging cells (11) that were grown to
50% confluence in IMDM supplemented with 5% FCS, 5% newborn calf
serum (HyClone), 10
g/ml protamine sulfate (Sigma-Aldrich, St. Quentin
Fallavier, France), IL-3, IL-6, and SCF. Control cells were cocultured on
⌿-CRIP or ⌿-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 Technol-
ogies) in the presence of GM-CSF (100 ng/ml; Schering-Plough, Levallois-
Perret, France) and 10% human serum AB (Centre National de la Trans-
fusion 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 ⫻10
5
/ml TCID50. Untransduced, neo-
transduced, or IFN-

-transduced macrophages were seeded in 6-well plates
at a concentration of 5 ⫻10
5
cells/ml and HIV-YU-2 was added for3hat
37°C and at a multiplicity of infection close to 0.01 in the presence of 10
g/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-

transgene integrations and
HIV DNA copies
The numbers of HIV DNA copies and IFN-

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 ob-
tained with the DNA preparations derived from plasmid-transfected U937
cells containing one copy of IFN-

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
␣
-globin set primer (18).
Quantification of cytokines by ELISA
The cytokines and chemokines IL-1
␣
, TNF-
␣
, IFN-
␥
, IL-12, MIP-1
␣
,
MIP-1

, and RANTES were quantified from cell-free supernatants of mac-
rophages 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
g 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
M
[
33
P]
␣
dCTP to detect the human glyceraldehyde-3-phosphate dehydroge-
nase transcripts as a quantitative control. To estimate chemokine receptor
expression, cDNA products were amplified for 40 cycles using the follow-
ing 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⬘-GGT
GTAATGAAGACCTTC-3⬘antisense. The reaction products were de-
tected by autoradiography after electrophoresis on 4% nondenaturing poly-
acrylamide gels and were quantified using the PhosphorImager (Molecular
Dynamics Sevenoaks, U.K.).
Results
Retrovirally mediated IFN-

transduction of macrophages
confers high anti-HIVYU-2 resistance
To obtain high numbers of IFN-

-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-K
b
HuIFN-

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 I). 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-

-
transduced macrophages secreted 480-1045 U/10
6
cells per 3 days
of IFN-

, whereas untransduced and neo-transduced cells pro-
duced no detectable levels of IFN-

(Table I and data not shown).
Table I. Phenotyping of untransduced (UT) and IFN-

-transduced (IFN-T) macrophages
a
Transduction
Efficacy (%) IFN production
(U/10
6
cells/72 h)
Cell Surface Markers Expression
CD4 CD14 HLA-ABC HLA-DR
MFI* % MFI % MFI % MFI %
AUT — ⬍60 8 6356753690 8070
IFN-T 50 480 8 58 48 74 50 97 145 75
BUT — ⬍60 6 68 37 75 ND 97 97 95
IFN-T 100 1045 8 63 37 71 ND 98 171 87
a
The percentage of transgene integrations, the IFN-

production, and the expression of cell-surface antigens were determined 1 mo after transduction. nd, not done; MFI,
mean of fluorescence.
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Up to 3 wk after gene transduction, the survival of IFN-

-produc-
ing macrophages was similar to that of untransduced or neo-trans-
duced 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 I). Expression of CD4 and CD14 was not mod-
ified by IFN-

transduction, whereas expression of MHC class I
and class II Ags was slightly increased (Table I). Moreover, both
untransduced and IFN-

-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-

had any apparent effect on cell differentiation.
To assess the in vitro efficacy of low constitutive expression of
IFN-

on HIV infection, IFN-

-transduced macrophages were
tested for resistance to M-cell-tropic HIV-YU-2 challenge. We ob-
served that untransduced and neo-transduced cells could be pro-
ductively infected by HIV-YU-2. As shown in Fig. 1, 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. 1, Cand D). In contrast, p24 Ag secretion by IFN-

-
transduced macrophages remained extremely low throughout the
culture in the 4 donors tested (Fig. 1). Furthermore, the infectivity
of HIV particles released by IFN-

-transduced macrophages 9
days after infection (determined on P4-2 Hela cells (20)) was re-
duced 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-

(Fig. 1D), further
confirming the specific ability of IFN-

to confer HIV resistance to
macrophages. These results were correlated with a drastic reduc-
tion of HIV DNA copy number per cell in IFN-

-transduced mac-
rophages compared with untransduced macrophages. As shown in
Fig. 2, 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-

-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-

-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-

transduction strongly
inhibits HIV-YU-2 and HIV-BAL infection of macrophages.
IFN-

transduction enhances Th1-type cytokine and

-
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

-chemokines was thus determined in neo-transduced and IFN-

-
transduced macrophages 9 days after the onset of HIV infection. A
similar amount (18 pg/10
6
cells) of IL-12 was detected in super-
natants from HIV-infected and uninfected macrophages (Fig. 3),
whereas there is a 10-fold increase of IFN-
␥
production by mac-
rophages after HIV infection. Moreover, in uninfected macro-
phages, the Th1-type cytokine production was enhanced after
IFN-

transduction. The IL-12 and IFN-
␥
production were 3-fold
and 14-fold higher, respectively, in IFN-

-transduced compared
with neo-transduced (Fig. 3) or untransduced macrophages (data
not shown). In IFN-

-transduced macrophages, similar levels of
IFN-
␥
were detected in uninfected and HIV-infected cells. The
production of TNF-
␣
and IL-1
␣
proinflammatory cytokines was
also analyzed in neo-transduced and IFN-

-transduced cells.
TNF-
␣
production was 23-fold higher after HIV infection, and
IL-1
␣
production, which was undetectable in uninfected cells
(⬍1.5 pg/10
6
cells), went up to 445 pg/10
6
cells in HIV-infected
cells. On the contrary, IFN-

transduction of the cells did not
modify the production of these proinflammatory cytokines in un-
infected or HIV-infected cells, confirming that in these cells HIV
replication was inhibited.
Several reports indicate that HIV infection of macrophages re-
sults in an increased production of the

-chemokines which are
ligands for the chemokine receptor CCR-5 (21, 22). Therefore, we
compared RANTES, MIP-1
␣
, and MIP-1

production in neo-
transduced and IFN-

-transduced macrophages after HIV infection.
The production of MIP-1
␣
and MIP-1

was increased 4-fold and
6-fold, respectively, by HIV infection (Fig. 3). IFN-

transduction
induced an enhanced production of these

-chemokines (6-fold,
FIGURE 1. Inhibition of HIV-YU-2 replication by IFN-

transduction
of macrophages. A–Drepresent the cells from four independent donors.
About 1 month after IFN-

transduction, macrophages were infected with
HIV. Cell culture supernatants from untransduced (UT), neo-transduced
(neo-T), or IFN-

-transduced (IFN-T) cells or from cells treated with 1000
U/ml of recombinant IFN-

(rec IFN) were collected at time points indi-
cated, and HIV p24 ELISA was performed.
FIGURE 2. IFN-

transduction of macrophages significantly reduces
the number of HIV-YU-2 DNA copies per cell. Aand Brepresent the cells
from two independent donors. About 1 month after IFN-

transduction,
macrophages were infected with HIV. DNA was extracted from untrans-
duced (UT) and IFN-

-transduced (IFN-T) cells at time points indicated, and
the number of HIV DNA copies per cell was determined by PCR analysis.
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-MEDIATED HIV RESISTANCE IN MACROPHAGES
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2-fold, and 4-fold for RANTES, MIP-1
␣
, and MIP-1

, respectively)
that was not modified after HIV infection of the cells, further con-
firming that the cells were resistant to HIV-YU-2 infection.
HIV resistance observed in IFN-

-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-

-transduced macrophages could be mediated by
RANTES because we have observed a significant increase of
RANTES production after IFN-

transduction of the cells. As
shown in Fig. 4, the addition of RANTES to neo-transduced mac-
rophages resulted in HIV resistance. Moreover, the addition of
blocking Ab to RANTES in IFN-

-transduced cell cultures abol-
ished the HIV-YU-2 resistance, indicating that RANTES is re-
quired for HIV resistance of IFN-

-transduced macrophages (Fig.
4). We then investigated the ability of IFN-

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. 5 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-

transduction of the cells, whereas we observed a 6-fold
reduction of transcripts for CCR-5 in IFN-

-transduced macro-
phages compared with untransduced macrophages (Fig. 5). Flow
cytometry analysis confirmed that a treatment of macrophages
with 1000 U/ml of IFN-

decreased cell surface expression of
CCR-5, whereas it had no effect on CXCR-4 expression (Fig. 6).
These results indicate that IFN-

-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.
Discussion
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 effi-
cient transduction of hematopoietic stem cells derived from HIV-
infected patients using an IFN-

-carrying retroviral vector will
FIGURE 3. IFN-

transduction of macrophages increases the produc-
tion of Th1-type cytokines and

-chemokines but not that of proinflam-
matory cytokines. IFN-
␥
and IL-12 Th1-type cytokines, IL-1
␣
and TNF-
␣
proinflammatory cytokines, and MIP-1
␣
, MIP-1

, and RANTES

-che-
mokines were quantified by ELISA in the culture medium of neo-trans-
duced (neo-T, gray histograms) or IFN-

-transduced macrophages (IFN-T,
black histograms) in uninfected (UI) and HIV-infected cells (HIV). These
results are representative of three independent experiments.
FIGURE 4. IFN-

transduction of macrophages significantly reduces
the number of HIV-YU-2 DNA copies per cell through RANTES produc-
tion. Neo-transduced (neo-T) and IFN-

-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, macro-
phages were treated with 10
g/ml of anti-RANTES mAb (aRANTES) or
with 10 ng/ml of recombinant RANTES.
FIGURE 5. IFN-

transduction of macrophages significantly reduces
CCR-5 expression. Nine days after HIV infection, RNA was extracted
from neo-transduced (neo-T) and IFN-

-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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require an extensive preparatory investigation. As one approach to
this aim, we stimulated CD34
⫹
cells for 15 days with a combina-
tion of cytokines before IFN-

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 dif-
ferentiation or expression of CD4 and CD14 cell surface Ags.
The ability of low constitutive expression of IFN-

to inhibit
viral replication in macrophages was then examined. Our data
showed that IFN-

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 neo-
transduced cells. This is consistent with our previous observations
that constitutive IFN-

production confers resistance against T-
tropic (HIV-BRU) and M-tropic (HIV-YU-2, HIV-BAL) in sev-
eral 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 infec-
tion in vitro (25–27).
In HIV-infected control macrophages, an increased secretion of
the proinflammatory cytokines TNF-
␣
and IL-1
␣
was oberved,
contrasting with HIV-infected IFN-

-transduced macrophages in
which the levels of IL-1
␣
and TNF-
␣
remained undetectable. Sim-
ilar observations have been made in IFN-

-transduced PBL from
HIV-infected patients (11). Moreover, elevated levels of proin-
flammatory cytokines were detected in the serum of HIV-infected
patients (28, 29). Because it has been shown that HIV-1 tat protein
induces TNF-
␣
, IL-1
␣
, and IFN-
␥
production (30–32), it is likely
that the undetectable level of IL-1
␣
and TNF-
␣
in IFN-

-trans-
duced macrophages reflects resistance to HIV infection.
High levels of production of proinflammatory cytokines are det-
rimental in the context of AIDS because they can alter immune
reponses, cause tissue damage, and up-regulate HIV replication
(33). IL-1 and TNF-
␣
are also involved in the pathogenic mech-
anisms of Kaposi sarcoma (34–36) and neurologic disease. Per-
sidsky et al. (37) suggested that the up-regulation of TNF-
␣
, IL-6,
and IL-10 is a major event that permits the transendothelial mi-
gration of monocytes into brain tissue, thus expanding the viral
reservoir in the brain (38) leading to progressive neurologic im-
pairment that appears at late stages of AIDS. Thus, IFN-

trans-
duction of macrophages may also be a therapeutic opportunity for
the prevention of AIDS-associated dementia because the levels of
proinflammatory cytokines in IFN-

-transduced macrophages are
not up-regulated after HIV infection.
We demonstrated that IFN-

-transduced macrophages secreted
3- and 14-fold more IL-12 and IFN-
␥
, respectively, compared with
untransduced cells. Increased production of Th1-type cytokines
was observed after IFN-

transduction of PBL (11) and dendritic
cells.
5
Type I IFN are known to increase the frequency of Th1 cells
(39–42). 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 re-
sponses (43, 44). Our results show that IFN-

transduction of mac-
rophages 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 cyto-
kines, we also observed that IFN-

transduction of macrophages
enhanced the secretion of the

-chemokines RANTES, MIP-1
␣
,
and MIP-1

. HIV infection of macrophages induces an up-regu-
lation of

-chemokine production, which has been reported by others
(21, 22). The enhanced release of

-chemokines in the tissues by
HIV-infected macrophages and by IFN-

transduction might attract
uninfected T cells and monocytes to the site of active infection.
Of the three

-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 in-
hibit HIV replication, recombinant RANTES was added before
HIV infection of macrophages. As shown in Fig. 5, 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-

-transduced macrophages
played a role in the inhibition of HIV replication. The addition of
RANTES-blocking Ab neutralized the inhibitory activity of IFN-

transduction of macrophages. Thus, our data suggest that the IFN-

-dependent release of RANTES by macrophages plays major role
in the inhibition of HIV replication. The simultaneous neutraliza-
tion of RANTES, MIP-1
␣
, and MIP-1

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-

transduction of CD34
⫹
TF-1 cells results in a protection
against HIV-YU-2 infection that is correlated with a 5-fold de-
crease in CCR-5 expression (14). CCR-5 expression was examined
in macrophages and revealed a 6-fold decrease of the mRNA tran-
scripts for CCR-5 in IFN-

-transduced cells compared with un-
transduced cells. FACS analysis revealed a decreased expression
of CCR-5 after a treatment of macrophages with recombinant
IFN-

. 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-
␣
inhibits
HIV replication in macrophages by inducing the production of
RANTES and by decreasing CCR-5 expression. These data
5
Cremer, I., V. Vieillard, C. Saute`s-Fridman, and E. De Maeyer. Inhibition of HIV
transmission to CD4
⫹
T cells after gene transfer of constitutively expressed IFN-

to
dendritic cells. Submitted for publication.
FIGURE 6. IFN-

treatment of macrophages reduces cell surface ex-
pression of CCR-5. Macrophages were treated with 1000 U/ml of recom-
binant IFN-

for 3 days. The levels of CCR-5 and CXCR-4 expression
were determined by FACS analysis. Solid lines represent untreated mac-
rophages, dotted lines represent IFN-

-treated macrophages, and bold lines
represent isotype control background staining.
1586 IFN-

-MEDIATED HIV RESISTANCE IN MACROPHAGES
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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-

transduction
of macrophages. Our data indicate that low constitutive production
of IFN-

can be used as an approach to somatic-cell gene therapy
of HIV infection, to inhibit viral replication, and to improve im-
mune functions.
Acknowledgments
We thank Catherine Saute`s-Fridman for critical reading of the manuscript
and the personnel of the maternity ward of the Orsay Hospital for providing
umbilical cord blood.
References
1. Gendelman, H. E., J. M. Orenstein, L. M. Baca, B. Weiser, H. Burger,
D. C. Kalter, and M. S. Meltzer. 1989. The macrophage in the persistence and
pathogenesis of HIV infection. AIDS 3:475.
2. Sierra-Madero, J. G., Z. Toossi, D. L. Hom, C. K. Finegan, E. Hoenig, and
E. A. Rich. 1994. Relationship between load of virus in alveolar macrophages
from human immunodeficiency virus type 1-infected persons, production of cy-
tokines, and clinical status. J. Infect. Dis. 169:18.
3. Meltzer, M. S., D. R. Skillman, P. J. Gomatos, D. C. Kalter, and
H. E. Gendelman. 1990. Role of mononuclear phagocytes in the pathogenesis of
human immunodeficiency virus infection. Annu. Rev. Immunol. 8:169.
4. Orenstein, J. M., C. Fox, and S. M. Wahl. 1997. Macrophages as a source of HIV
during opportunistic infections. Science 276:1857.
5. Baldwin, G. C., J. Fleischmann, Y. Chung, Y. Koyanagi, I. S. Chen, and
D. W. Golde. 1990. Human immunodeficiency virus causes mononuclear phago-
cyte dysfunction. Proc. Natl. Acad. Sci. USA 87:3933.
6. Pantaleo, G. 1997. How immune-based interventions can change HIV therapy.
Nat. Med. 3:483.
7. De Maeyer, E., and J. De Maeyer-Guignard. 1988. Interferons and Other Reg-
ulatory Cytokines. Wiley, New York.
8. Francis, M. L., M. S. Meltzer, and H. E. Gendelman. 1992. Interferons in the
persistence, pathogenesis, and treatment of HIV infection. AIDS Res. Hum. Ret-
roviruses 8:199.
9. Michaelis, B., and J. A. Levy. 1989. HIV replication can be blocked by recom-
binant human interferon

.AIDS 3:27.
10. Vieillard, V., E. Lauret, V. Rousseau, and E. De Maeyer. 1994. Blocking of
retroviral infection at a step prior to reverse transcription in cells transformed to
constitutively express interferon

.Proc. Natl. Acad. Sci. USA 91:2689.
11. Vieillard, V., I. Cremer, E. Lauret, W. Rozenbaum, P. Debre, B. Autran, and
E. De Maeyer. 1997. Interferon

transduction of peripheral blood lymphocytes
from HIV-infected donors increases Th1-type cytokine production and improves
the proliferative response to recall antigens. Proc. Natl. Acad. Sci. USA 94:11595.
12. Danos, O., and R. C. Mulligan. 1988. Safe and efficient generation of recombi-
nant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad.
Sci. USA 85:6460.
13. Hawley, R. G., L. A. Sabourin, and T. S. Hawley. 1989. An improved retroviral
vector for gene transfer into undifferentiated cells. Nucleic Acids Res. 17:4001.
14. Cremer, I., V. Vieillard, and E. De Maeyer. 1999. Interferon-

-induced human
immunodeficiency virus resistance in CD34
⫹
human hematopoietic progenitor
cells: correlation with a down-regulation of CCR-5 expression. Virology 253:241.
15. Li, Y., J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn.
1991. Molecular characterization of human immunodeficiency virus type 1
cloned directly from uncultured human brain tissue: identification of replication-
competent and -defective viral genomes. J. Virol. 65:3973.
16. Mace, K., I. Seif, C. Anjard, J. De Maeyer-Guignard, M. D. Dodon, L. Gazzolo,
and E. De Maeyer. 1991. Enhanced resistance to HIV-1 replication in U937 cells
stably transfected with the human IFN-

gene behind an MHC promoter frag-
ment. J. Immunol. 147:3553.
17. Vieillard, V., E. Lauret, V. Maguer, C. Jacomet, W. Rozenbaum, L. Gazzolo, and
E. De Maeyer. 1995. Autocrine interferon-

synthesis for gene therapy of HIV
infection: increased resistance to HIV-1 in lymphocytes from healthy and HIV-
infected individuals. AIDS 9:1221.
18. Erhart, M., A. K. Piller, and S. Weaver. 1987. Polymorphism and gene conver-
sion in mouse
␣
-globin haplotypes. Genetics 115:511.
19. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156.
20. Charneau, P., G. Mirambeau, P. Roux, S. Paulous, H. Buc, and F. Clavel. 1994.
HIV-1 reverse transcription: a termination step at the center of the genome.
J. Mol. Biol. 241:651.
21. Canque, B., M. Rosenzwajg, A. Gey, E. Tartour, W. H. Fridman, and
J. C. Gluckman. 1996. Macrophage inflammatory protein-1
␣
is induced by hu-
man immunodeficiency virus infection of monocyte-derived macrophages. Blood
87:2011.
22. Schmidtmayerova, H., H. S. Nottet, G. Nuovo, T. Raabe, C. R. Flanagan,
L. Dubrovsky, H. E. Gendelman, A. Cerami, M. Bukrinsky, and B. Sherry. 1996.
Human immunodeficiency virus type 1 infection alters chemokine

peptide ex-
pression in human monocytes: implications for recruitment of leukocytes into
brain and lymph nodes. Proc. Natl. Acad. Sci. USA 93:700.
23. Miller, D. G., M. A. Adam, and A. D. Miller. 1990. Gene transfer by retrovirus
vectors occurs only in cells that are actively replicating at the time of infection.
Mol. Cell. Biol. 10:4239.
24. Gessani, S., P. Puddu, B. Varano, P. Borghi, L. Conti, L. Fantuzzi, and
F. Belardelli. 1994. Induction of

interferon by human immunodeficiency virus
type 1 and its gp120 protein in human monocytes-macrophages: role of

inter-
feron in restriction of virus replication. J. Virol. 68:1983.
25. Rosenzweig, M., D. F. Marks, D. Hempel, J. Lisziewicz, and R. P. Johnson. 1997.
Transduction of CD34
⫹
hematopoietic progenitor cells with an antitat gene pro-
tects T-cell and macrophage progeny from AIDS virus infection. J. Virol. 71:
2740.
26. Bonyhadi, M. L., K. Moss, A. Voytovich, J. Auten, C. Kalfoglou, I. Plavec,
S. Forestell, L. Su, E. Bohnlein, and H. Kaneshima. 1997. RevM10-expressing T
cells derived in vivo from transduced human hematopoietic stem-progenitor cells
inhibit human immunodeficiency virus replication. J. Virol. 71:4707.
27. Yu, M., M. C. Leavitt, M. Maruyama, O. Yamada, D. Young, A. D. Ho, and
F. Wong-Staal. 1995. Intracellular immunization of human fetal cord blood stem/
progenitor cells with a ribozyme against human immunodeficiency virus type 1.
Proc. Natl. Acad. Sci. USA 92:699.
28. Breen, E. C., A. R. Rezai, K. Nakajima, G. N. Beall, R. T. Mitsuyasu, T. Hirano,
T. Kishimoto, and O. Martinez-Maza. 1990. Infection with HIV is associated
with elevated IL-6 levels and production. J. Immunol. 144:480.
29. Arditi, M., W. Kabat, and R. Yogev. 1991. Serum tumor necrosis factor
␣
, in-
terleukin 1-

, p24 antigen concentrations and CD4
⫹
cells at various stages of human
immunodeficiency virus 1 infection in children. Pediatr. Infect. Dis. J. 10:450.
30. Rautonen, J., N. Rautonen, N. L. Martin, and D. W. Wara. 1994. HIV type 1 Tat
protein induces immunoglobulin and interleukin 6 synthesis by uninfected pe-
ripheral blood mononuclear cells. AIDS Res. Hum. Retroviruses 10:781.
31. Buonaguro, L., G. Barillari, H. K. Chang, C. A. Bohan, V. Kao, R. Morgan,
R. C. Gallo, and B. Ensoli. 1992. Effects of the human immunodeficiency virus
type 1 Tat protein on the expression of inflammatory cytokines. J. Virol. 66:7159.
32. Esser, R., W. Glienke, H. von Briesen, H. Rubsamen-Waigmann, and
R. Andreesen. 1996. Differential regulation of proinflammatory and hematopoi-
etic cytokines in human macrophages after infection with human immunodefi-
ciency virus. Blood 88:3474.
33. Vicenzi, E., and G. Poli. 1994. Regulation of HIV expression by viral genes and
cytokines. J. Leukocyte Biol. 56:328.
34. Birx, D. L., R. R. Redfield, K. Tencer, A. Fowler, D. S. Burke, and G. Tosato.
1990. Induction of interleukin-6 during human immunodeficiency virus infection.
Blood 76:2303.
35. Ensoli, B., S. Nakamura, S. Z. Salahuddin, P. Biberfeld, L. Larsson, B. Beaver,
F. Wong-Staal, and R. C. Gallo. 1989. AIDS-Kaposi’s sarcoma-derived cells
express cytokines with autocrine and paracrine growth effects. Science 243:223.
36. Miles, S. A., A. R. Rezai, J. F. Salazar-Gonzalez, M. Vander Meyden,
R. H. Stevens, D. M. Logan, R. T. Mitsuyasu, T. Taga, T. Hirano, T. Kishimoto,
et al. 1990. AIDS Kaposi sarcoma-derived cells produce and respond to inter-
leukin 6. Proc. Natl. Acad. Sci. USA 87:4068.
37. Persidsky, Y., M. Stins, D. Way, M. H. Witte, M. Weinand, K. S. Kim, P. Bock,
H. E. Gendelman, and M. Fiala. 1997. A model for monocyte migration through
the blood-brain barrier during HIV-1 encephalitis. J. Immunol. 158:3499.
38. Price, R. W., B. Brew, J. Sidtis, M. Rosenblum, A. C. Scheck, and P. Cleary.
1988. The brain in AIDS: central nervous system HIV-1 infection and AIDS
dementia complex. Science 239:586.
39. Brinkmann, V., T. Geiger, S. Alkan, and C. H. Heusser. 1993. Interferon
␣
increases the frequency of interferon
␥
-producing human CD4
⫹
T cells. J. Exp.
Med. 178:1655.
40. Parronchi, P., M. De Carli, R. Manetti, C. Simonelli, S. Sampognaro,
M. P. Piccinni, D. Macchia, E. Maggi, G. Del Prete, and S. Romagnani. 1992.
IL-4 and IFN (
␣
and
␥
) exert opposite regulatory effects on the development of
cytolytic potential by Th1 or Th2 human T cell clones. J. Immunol. 149:2977.
41. McRae, B. L., L. J. Picker, and G. A. van Seventer. 1997. Human recombinant
interferon-

influences T helper subset differentiation by regulating cytokine se-
cretion pattern and expression of homing receptors. Eur. J. Immunol. 27:2650.
42. Wenner, C. A., M. L. Guler, S. E. Macatonia, A. O’Garra, and K. M. Murphy.
1996. Roles of IFN-
␥
and IFN-
␣
in IL-12-induced T helper cell-1 development.
J. Immunol. 156:1442.
43. Clerici, M., and G. M. Shearer. 1994. The Th1-Th2 hypothesis of HIV infection:
new insights. Immunol. Today 15:575.
44. Clerici, M., L. De Palma, E. Roilides, R. Baker, and G. M. Shearer. 1993. Anal-
ysis of T helper and antigen-presenting cell functions in cord blood and peripheral
blood leukocytes from healthy children of different ages. J. Clin. Invest. 91:2829.
45. Verani, A., G. Scarlatti, M. Comar, E. Tresoldi, S. Polo, M. Giacca, P. Lusso,
A. G. Siccardi, and D. Vercelli. 1997. C-C chemokines released by lipopolysac-
charide (LPS)-stimulated human macrophages suppress HIV-1 infection in both
macrophages and T cells. J. Exp. Med. 185:805.
46. Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, and
P. Lusso. 1995. Identification of RANTES, MIP-1
␣
, and MIP-1

as the major
HIV-suppressive factors produced by CD8
⫹
T cells. Science 270:1811.
47. Wang, J., G. Roderiquez, T. Oravecz, and M. A. Norcross. 1998. Cytokine regulation
of human immunodeficiency virus type 1 entry and replication in human monocytes/
macrophages through modulation of CCR5 expression. J. Virol. 72:7642.
48. Lane, B. D., M. M. Markovitz, N. L. Woodford, R. Rochford, R. M. Strieter, and
M. J. Coffey. 1999. TNF-
␣
inhibits HIV-1 replication in peripheral blood mono-
cytes and alveolar macrophages by inducing the production of RANTES and
decreasing C-C chemokine receptor (CCR5) expression. J. Immunol. 163:3653.
1587The Journal of Immunology
by guest on January 25, 2017http://www.jimmunol.org/Downloaded from
