Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada
Correspondence: Michel J. Tremblay, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: Michel.J.Tremblay{at}crchul.ulaval.ca
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B. Mono Mac 1 can thus be considered as a human
monocytoid cell line representing a proper in vitro system
for studying the complex interactions between HIV-1 and cells of the
monocyte/macrophage lineage.
Key Words: monocytes/macrophages • AIDS/HIV • cellular differentiation • cell surface molecules • transcription factors
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Primary human monocyte/macrophages have been extensively used to define the role played by cells of the mononuclear phagocyte lineage in the context of HIV-1 infection. Unfortunately, major problems have been reported in in vitro studies when using such cells as a target for HIV-1 infection. For example, marked differences in the ability to support a productive HIV-1 infection were seen depending on several variables such as the purity of the isolated population, the maturation state of the cell (length of time in culture), and the degree of macrophage adherence [10 ]. The donor source represents another confounding factor influencing the magnitude of HIV-1 replication. Promonocytic cell lines and primary monocyte/macrophages display some similarities with respect to expression of surface markers (e.g., CD14, MHC-II) and functional capacities (e.g., antigen presentation, accessory cell functions). This has prompted several investigators to use human myeloid leukemic cell lines as models to examine the ability of cells of the monocyte/macrophage lineage to support productive infection with monocytotropic strains of HIV-1. The most frequently used immortalized monocytoid cell lines to study HIV-1 infection in vitro remain the leukemia cell lines U937, THP-1, and HL-60 [11 12 13 ]. It should be stated that, in contrast to primary monocyte-derived macrophages (MDM), these monocytic cell lines once differentiated to a macrophage phenotype, by means of agents such as phorbol 12-myristate 13-acetate (PMA) or bacterial lipopolysaccharide (LPS), display a low permissiveness to infection by M-tropic isolates of HIV-1 [14 15 16 17 ]. Therefore, differentiated monocytoid cell lines show a very different pattern of susceptibility to infection by various M-tropic HIV-1 strains and do not reflect their corresponding primary cell types in studies focused on viral host cell tropism [14 , 15 , 18 19 20 21 ].
The established human tumor cell line Mono Mac 1 has recently been
defined as a good model system to study hematological and immunological
properties of cells from a monocytoid origin because this cell line
displays morphological, histochemical, phenotypic, as well as
functional properties of mature monocytes (e.g., phagocytosis, Fc
receptor, and specific surface marker expression) [22
,
23
]. Furthermore, after LPS treatment, which has been
reported to induce a macrophage-like phenotype, the Mono Mac 1 cell
line also presents phenotypic and physiological properties such as
cytokine secretion [tumor necrosis factor 
(TNF-),
interleukin-1ß (IL-1ß), granulocyte colony-stimulating factor
(G-CSF), and granulocyte-macrophage CSF (GM-CSF)] and specific antigen
presentation (CD14), which resemble mature MDMs [22
,
23
]. Given that U937, THP-1, and HL-60 are considered
either as cells at early stages of monocytic differentiation (U937 and
THP-1) or are clearly myeloid (HL-60) and are poorly susceptible to
infection with M-tropic viral strains, we tested the susceptibility of
the human cell line Mono Mac 1 to HIV-1 infection. In this study, we
provide evidence that Mono Mac 1 represents an appropriate model system
that parallels primary MDM properties in terms of necessary surface
receptor/coreceptors mediating HIV-1 infection by both M- and
dual-tropic strains of HIV-1, infection kinetics, and cellular
differentiation events. Therefore, Mono Mac 1 can be considered as a
representative biological system for in vitro studies of
HIV-1 infection in cells of the monocyte/macrophage lineage.
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Flow cytometric analysis
Untreated (monocyte-like) and LPS-treated (macrophage-like) Mono
Mac 1 cells (5 x 105) were incubated for 30 min on
ice with saturating concentrations (1 µg/106 cells) of
monoclonal antibodies directed against CD4 (clone SIM.4), CCR5 (clone
2D7), CXCR4 (clone 12G5), CCR3 (clone 7B11), and CD14 (clone CRIS-6).
Cells were next incubated for 30 min on ice with a saturating
concentration of a secondary antibody constituted of
R-phycoerythrin-conjugated goat anti-mouse immunoglobulin G (IgG) from
Caltag Laboratories (San Francisco, CA). Finally, cells were
resuspended in 500 µL of phosphate-buffered saline (PBS) containing
1% (w/v) paraformaldehyde before flow cytometry analysis (EPICS XL,
Coulter, Miami, FL). Controls consisted of commercial isotype-matched
irrelevant murine monoclonal antibodies (Sigma). Anti-CD4 (SIM.4) and
anti-CCR3 (7B11) monoclonal antibodies were kindly supplied by the AIDS
Research and Reference Reagent Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases. Anti-CXCR4 (12G5) and
anti-CCR5 (2D7) monoclonal antibody were purchased from PharMingen,
while anti-CD14 (CRIS-6) was obtained from Biosource International.
Plasmids and preparation of virus stocks
The NL4-3 luciferase backbone
(pNL4-3-Luc-E-R+) and pcDNA-I/Amp-based
expression vectors coding for HIV-1 ADA (R5), JR-FL (R5), BaL (R5),
HXB2 (X4), or amphotropic murine leukemia virus (A-MLV) full-length
envelope proteins were generously provided by Nathaniel R. Landau (The
Salk Institute for Biological Studies, La Jolla, CA)
[9
]. The infectious molecular clones pNL4-3 (X4)
[24
] and p89.6 (R5X4) [25
] were also used
in this study. pNL4-3 was provided by the AIDS Repository Program,
whereas pcDNA-I/89.6 was obtained from Ronald Collman (Pulmonary and
Critical Care Division, University of Pennsylvania, Philadelphia, PA).
The ADA macrophage-tropic virions [26
] were prepared by
infecting primary human MDM. The ADA strain of HIV-1 was kindly
provided by Howard E. Gendelman, as cell-free supernatant from infected
MDM, through the AIDS Repository Program. The pHCMV-G expressing the
broad-host-range vesicular stomatitis virus envelope glycoprotein G
(VSV-G) from the human cytomegalovirus (HCMV) promoter has been
described previously [27
]. Fully infectious viral
entities (NL4-3, ADA, 89.6) and luciferase reporter viruses pseudotyped
with various envelope proteins (ADA, JR-FL, BaL, HXB2, A-MLV, VSV-G)
were generated by calcium phosphate cotransfection in 293T cells as
described previously [28
, 29
]. Negative
controls for experiments conducted with luciferase-encoding virions
consisted of envelope-free viruses. Virus-containing supernatants were
harvested 40 h posttransfection and frozen at -85°C until used.
Virus preparations were quantified using a commercial assay for the
viral major core protein p24 (Organon Teknika, Durham, NC).
Viral infection assay
Untreated (monocyte-like) and LPS-treated
(macrophage-like) Mono Mac 1 cells (1 x 105) were
seeded in 96-well dishes in complete culture medium and infected with
luciferase-encoding pseudotyped virions (10 ng of p24) in a total
volume of 200 µL. After 72 h, 100 µL of cell-free supernatant
from each well were removed and complemented with 25 µL of 5x
Promega cell culture lysis buffer [125 mM triphosphate (pH 7.8), 10 mM
dithiothreitol (DTT), 5% Triton X-100, 50% glycerol] for a 30-min
incubation period at room temperature. An aliquot of 20 µL from the
lysate was then mixed with 100 µL of luciferase assay buffer [210 mM
tricine, 1.07 mM (MgCO3)4
Mg(OH)2• 5H2O, 2.67 mM MgSO4, 0.1
mM EDTA, 270 µM CoA, 470 µM luciferine, 530 µM ATP, 33.3 mM
DTT]. Finally, luciferase activity was monitored using a microplate
luminometer (MLX; Dynex Technologies, Chantilly, VA). Kinetics of viral
infection was assessed by infecting untreated (monocyte-like) and
LPS-treated (macrophage-like) Mono Mac 1 cells (3 x
105) in 24-well dishes (final volume of 500 µL) with
fully infectious progeny viruses (NL4-3, ADA, 89.6/30 ng of
p24). Cells were incubated for 25–32 days, during which aliquots of
100 µL of cell-free supernatant were retrieved to evaluate reverse
transcriptase activity as described previously [30
].
Virally infected Mono Mac 1 cells were observed in light microscopy for
the presence of HIV-1-mediated syncytium formation and were
photographed at a magnification of x100 with an inverted microscope.
Viral entry assay
Undifferentiated Mono Mac 1 cells (1 x 106
cells/mL, 250 µL/well) were exposed to similar amounts of
HIV-1NL4-3 (X4) or HIV-1JR-FL (R5) (100 ng of
p24) in complete culture medium for 2.5 h at 37°C. Cells were
washed twice with 250 µL of ice-cold PBS and were next resuspended in
250 µL of cold RPMI 1640 (without FBS) containing pronase (Boehringer
Mannheim, Laval, Quebec) at 0.1 mg/mL for 5 min at 4°C. Cells were
washed immediately with 2 mL of ice-cold RPMI 10 containing 10% FBS
and three times with ice-cold PBS to eliminate pronase. Cells were
resuspended in 1 mL of complete culture medium to which was added 200
µL of disruption buffer (0.5% Triton X-100 in PBST). Cells were
agitated for 10 min at room temperature and then stored at -20°C
until assayed for p24 content.
Semiquantitative PCR analysis
After LPS treatment and infection with HIV-1, total cellular DNA
was isolated using a DNA extraction kit (Quiagen). The PCR reaction
mixture was made of 1.5 µg of DNA, 200 µM of each four
deoxynucleotide triphosphates along with MgCl2 (1x), Taq
buffer (1x), and 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus).
This reaction mixture was covered with 40 µL of mineral oil and then
subjected to denaturation (10 min at 94°C) followed by 27 cycles of 1
min at 94°C, 2 min at 56°C, 3 min at 72°C, and finally 10 min at
72°C for complete polymerization. Primer pairs used for these
experiments included M667/M661, which can detect full-length or nearly
completely synthesized HIV-1 DNA (200-bp fragment) [31
].
In parallel, the primer set 14-33/123-104 was used to amplify a 100-bp
fragment specific for the human ß-globin gene.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared according to the previously
described microscale preparation protocol [32
]. Briefly,
untreated and LPS-treated Mono Mac 1 cells (5 x 106)
were washed with ice-cold PBS and resuspended in 400 µL of cold
buffer A [10 mM
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
acid (HEPES; pH7.9), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and
0.5 mM phenylmethylsulfonyl fluoride(PMSF)]. The cells were allowed to
swell on ice for 15 min, after which 25 µL of a 10% solution of
Nonidet P-40 is added and the tube is vigorously vortexed for 30 s. The homogenate was centrifuged for 10 s at 12,000 g.
The supernatant fraction was discarded and the pellet was resuspended
in 50 µL of cold buffer C [20 mM HEPES-KOH (pH 7.9), 0.4 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF] and incubated at 4°C
for 15 min on a shaking platform. Cellular debris were removed by
centrifugation at 12,000 g for 20 min at 4°C and the
supernatant fractions were stored at -70°C until used. Ten
micrograms of nuclear extracts were used to perform EMSA as determined
by the bicinchoninic acid assay (BCA) with a commercial protein assay
reagent (Pierce, Rockford, IL). Nuclear extracts were incubated for 20
min at room temperature in 15 µL of the binding buffer [100 mM HEPES
(pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol,
5 mM EDTA, 250 mM NaCl, 2 µg poly (dI-dC), 10 µg nuclease-free
bovine serum albumin fraction V] containing 1 ng of
32P-5’-end-labeled double-stranded (dsDNA)
oligonucleotide. Double-stranded DNA (100 ng) was labeled with
[
-32P]ATP and T4 polynucleotide kinase in a kinase
buffer (New England Biolabs, Beverly, MA). This mixture was incubated
for 30 min at 37°C and the reaction was stopped with 5 µL of 0.2 M
EDTA. The labeled oligonucleotide was extracted with phenol/chloroform
and passed through a G-50 spin column. The dsDNA oligonucleotide, which
was used as a probe, contained the consensus NF-B and HIV-1
enhancer-binding site corresponding to the sequences
5’-ATGTGAGGGGACTTTCCCAGGC-3’ and
5’-CAAGGGACTTTCCGCTGGGGACTTTCCAGGG-3, respectively. The DNA-protein
complexes were migrated on a 4% (w/v) polyacrylamide gel containing
0.5xTris-Borate-EDTA. The gels were then subsequently dried and
autoradiographed. Specific DNA-protein complexes were identified
through the use of 100-fold competitions of cold specific DNA
oligonucleotides.
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Next, in order to evaluate the functional capacity of Mono Mac 1 to act as a cellular target for HIV-1 infection, we measured by flow cytometry the levels of HIV-1 primary receptor and coreceptors at the cell surface. The cell-surface markers CD4, CCR5, CXCR4, and CCR3 were expressed on 58, 100, 73, and 40%, respectively, of untreated Mono Mac 1 cells (Fig. 1 , left panels). It is interesting to note that, as it is the case for Mono Mac 1 cells, primary human MDM have also been reported to be positive for CD4 and to express detectable levels of CXCR4 on their membranes [33 ]. Treatment of Mono Mac 1 cells with 10 ng/mL of LPS for 72 h induces a slight decrease in the levels of surface expression of CD4, CXCR4, and CCR3 (Fig. 1 , right panels). However, LPS-treatment of Mono Mac 1 slightly increases the intensity of CCR5 staining from a specific mean fluorescence intensity of 15.3 in undifferentiated cells (monocyte-like) to 17.7 for differentiated cells (macrophage-like). Maturation of Mono Mac 1 cells was confirmed by the observation that LPS treatment induces an increased adherence to the plastic surface of the culture vessels (data not shown). Differentiation of Mono Mac 1 was further distinguished by morphological changes of the cells because treatment with LPS resulted in the development of irregular shapes and more extensive projections of cell membranes (data not shown). These results suggest that LPS-mediated maturation of Mono Mac 1 cells from an immature (monocyte-like) to a more mature phenotype (macrophage-like) mimics the primary human monocyte/macrophage lineage.
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Figure 1. Flow cytometric studies of HIV-1 primary cellular receptor and
coreceptors. Mono Mac 1 cells were either left untreated or were
treated for 72 h with LPS (10 ng/mL) before staining with anti-CD4
(clone SIM.4), anti-CCR5 (clone 2D7), anti-CXCR4 (clone 12G5), and
anti-CCR3 (clone 7B11) monoclonal antibodies (dotted lines). Controls
consisted of cells incubated with an isotype-matched irrelevant
monoclonal antibody (solid lines).
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Figure 2. Single-round infection assays with recombinant luciferase-encoding
virions. (A) Untreated (open bars) and LPS-treated (filled bars) Mono
Mac 1 cells (1 x 105) were infected with HIV-1-based
luciferase reporter viruses (10 ng of p24) bearing either T- (HXB2) or
M-tropic (BaL, ADA, JR-FL) envelope proteins. (B) WE17/10 cells (1 x 105) were infected with HIV-1-based luciferase reporter
viruses (10 ng of p24) bearing either T- (HXB2) or M-tropic (BaL)
envelope proteins. Infection was allowed to proceed for 72 h
before lysis of the samples. Viral infections were translated in
luciferase activity that was read with a luminometer apparatus. Results
are shown as the mean ± SD of samples carried out in
triplicate. These data are representative of three independent
experiments. Controls consisted of cells infected with Env-deficient
luciferase-encoding viruses.
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Kinetics studies of HIV-1 infection
Our next interest was to assess the capacity of this human
monocytic cell line to support replication of unmodified complete
virions because HIV-1 replication is known to be a complex process that
is regulated by proteins of viral and cellular origin that has been
shown to work in cis and in trans. This goal was
achieved by infecting both untreated (monocyte-like) and LPS-treated
Mono Mac 1 cells (macrophage-like) with fully infectious viruses
displaying a tropism for either CD4+ T lymphocytes
(HIV-1NL4-3), monocyte/macrophages (HIV-1ADA),
or both of them (HIV-189.6). In this set of experiments,
the extent of virus replication was investigated by measuring reverse
transcriptase activity in cell-free culture supernatants. In accord
with our previous infection studies with T-tropic recombinant virions,
a weak virus production was detected at a very late time point in
untreated and LPS-treated Mono Mac 1 cells infected with the
CXCR4-dependent T cell line-adapted (TCLA) strain of HIV-1, NL4-3
(Fig. 3A
). In contrast, a sustained and much stronger virus production was
observed in undifferentiated Mono Mac 1 cells infected with the
M-tropic ADA isolate and the dual-tropic 89.6 strain of HIV-1 (Fig. 3B
3C)
. Again, virus production in cells infected with M-tropic isolates
was enhanced upon treatment of Mono Mac 1 with LPS, which confirms our
previous data with HIV-1-based luciferase reporter viruses.
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Figure 3. Kinetics of virus infection with fully infectious HIV-1 particles.
Untreated (open circles) and LPS-treated Mono Mac 1 cells (filled
circles; 3 x 105) were infected with fully infectious
strains of HIV-1 (NL4-3/T-tropic, ADA/M-tropic, and 89.6/dual-tropic;
30 ng of p24). Virus production was monitored by measuring reverse
transcriptase activity in cell-free supernatants at the indicated time
points. Data shown as the mean of triplicate samples and are
representative of two independent experiments.
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Table 1. Internalization of X4 and R5 Strains of HIV-1 in Undifferentiated Mono
Mac 1 Cells
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B
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Figure 4. Semiquantitative PCR analysis of viral DNA levels. (A) Untreated (lanes
2 and 4) and LPS-treated Mono Mac 1 cells (lanes 3 and 5) were either
left uninfected (lanes 2 and 3) or were infected with
HIV-1BaL (lanes 4 and 5) and cultured for 6 h before
DNA extraction. Two sets of primers were included in each tube: an
HIV-1 specific primer pair (M667/M661) and an oligonucleotide primer
pair specific for human ß-globin, which serves as an internal
control. Ratios between amplified signal with the HIV-1 specific set of
primers and the ß-globin set were calculated for each experimental
condition using an -imager spot density calculator. (B) Sensitivity
of the current semiquantitive PCR was evaluated through the use of
serial plasmid dilutions with the NL4-3 viral clone and the specific
primer pair M667/M661. Again, PCR products were calculated for each
template dilution using an -imager spot density calculator.
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B has been shown to
act as a major constituent in HIV-1 LTR-driven regulation and to be
essential for efficient HIV-1 expression [36
37
38
].
Reports have also indicated that nuclear NF-B is constitutively
expressed in cells of the monocyte/macrophage lineage (i.e., monocytic
cell lines, blood monocytes, tissue macrophages)
[38
39
40
41
]. Moreover, constitutive NF-B can be further
increased with stage of maturation [38
]. As illustrated
in Figures 2
and 3
, replication of M- and dual-tropic HIV-1 strains was
more efficient in LPS-treated than untreated Mono Mac 1 cells. In an
attempt to define whether the observed up-regulation in virus
production could be related to an increase in nuclear translocation of
NF-B, we conducted virus infection assays with luciferase reporter
viruses bearing either the amphotropic murine leukemia virus (A-MLV)
envelope protein or vesicular stomatitis virus envelope glycoprotein G
(VSV-G). Virions pseudotyped with A-MLV and VSV-G Env proteins will
enter Mono Mac 1 cells in a CD4/CCR5/CXCR4-independent manner. Higher
levels of virus-encoded reporter gene activity was still seen in
differentiated Mono Mac 1 cells compared with untreated cells after
infection with A-MLV and VSV-G pseudotyped recombinant viruses
(Fig. 5
). Data from this set of experiments suggest that the intracellular
milieu prevailing inside treated Mono Mac 1 cells is more favorable for
HIV-1 replication than in untreated cells. The role played by NF-B
in the increase of virus replication was assessed by performing gel
mobility shift assays in cell lysates before and after LPS treatment.
After treatment of Mono Mac 1 cells with LPS, a noticeable increase in
endogenous nuclear NF-B levels was detected when using a
radiolabeled probe corresponding to the consensus NF-B binding
sequence (Fig. 6A
). Given that the crucial role played by NF-B in up-regulation
of HIV-1 transcriptional activity is due to its association to the
HIV-1 LTR enhancer region [42
43
44
], we next performed
EMSA using a radiolabeled probe containing the complete HIV-1
enhancer-binding sequence. Again, binding of NF-B to the HIV-1 LTR
enhancer domain was increased upon treatment of Mono Mac 1 cells with
LPS (Fig. 6B) . Altogether, results from these studies suggest that the
increase in virus production that is seen in more mature Mono Mac 1
cells (macrophage-like) compared with Mono Mac 1 cells displaying a
more immature phenotype (monocyte-like) is most likely due to higher
levels of endogenous nuclear NF-B.
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Figure 5. Single-round infection experiments with CD4/CCR5/CXCR4-independent
reporter gene-encoding progeny viruses. Untreated (open bars) and
LPS-treated (filled bars) Mono Mac 1 cells (1 x 105)
were infected with HIV-1-based luciferase reporter viruses (10 ng of
p24) pseudotyped with A-MLV and VSV-G Envs. Samples were lysed after a
72-h incubation period and read for luciferase activity with a
luminometer. Results are shown as the mean ± SD of
samples carried out in triplicate. These data are representative of
three independent experiments. Controls consisted of cells infected
with Env-deficient luciferase-encoding viruses.
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Figure 6. Comparison of NF-B binding activity in untreated and LPS-treated
Mono Mac 1 cells. Nuclear extracts from untreated (NT) and LPS-treated
Mono Mac 1 cells (5 x 106) were extracted at 1, 2,
and 3 days after LPS treatment. Probes corresponding to the consensus
NF-B binding domain (A) and HIV-1 enhancer sequence (B) were used to
determine specific DNA-protein interactions by EMSA. Specific
DNA-protein complexes were identified using 100-fold competitions of
cold specific DNA oligonucleotides.
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Figure 7. Virus-mediated syncytium formation by M- and dual-tropic HIV-1 strains.
Undifferentiated (A) and LPS-treated Mono Mac 1 cells (B, C, and D)
were either left uninfected (A, B) or were infected with
fully-infectious ADA (M-tropic; panel C) and 89.6 (dual-tropic; panel
D). Cells were analyzed for HIV-1-mediated syncytium formation and
photographed at a magnification of x100 with an inverted microscope.
Arrows indicate syncytia.
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As specified above, previous reports have demonstrated that the most
widely used human cell lines of monocytoid origin do not represent an
ideal system to investigate the effects of HIV-1 infection on cells of
the monocyte/macrophage lineage [14
, 15
,
17
, 20
, 51
, 52
].
For example, Valentin and colleagues discovered that the M-tropic
isolate HIV-1BaL was unable to establish a productive
infection in any of the five human monocytoid cell lines tested (U937,
THP-1, RC2A, Mono Mac 6, and DD) [20
]. It was thus
concluded that these human continuous cell lines do not parallel
primary MDM, at least with respect to susceptibility to infection with
M-tropic HIV-1 variants. With this information in mind, we focused our
attention on Mono Mac 1, a recently established human cell line showing
characteristics of mature blood monocytes. This line has already been
extensively characterized in terms of reactive oxygen intermediates and
lysozyme production, phagocytosis, surface expression of specific
markers (e.g., CD13, CD14, CD15, CD68, MHC class II), and cytokine
production in response to LPS or PMA treatment (e.g., TNF-, G-CSF,
M-CSF, GM-CSF, IL-1ß) [22
, 23
].
We first proceeded to the immunophenotyping of Mono Mac 1 cells using flow microfluorimetry. We report here that Mono Mac 1 are expressing CD14, a marker found on cells committed to myeloid/monocytic lineages, and are also positive for CD4, the primary cellular receptor for HIV-1. It is interesting to note that our flow cytometric studies revealed that Mono Mac 1 cells also express the chemokine coreceptors CXCR4, CCR5, and CCR3. Differentiation of Mono Mac 1 to a more mature phenotype (macrophage-like) by treatment with bacterial-derived LPS resulted in a slight reduction of CD4, CXCR4, and CCR3 surface markers. In contrast, CCR5 antigen expression increased after LPS treatment, which is in agreement with a recent report showing that CCR5 mRNA level is up-regulated in PMA-treated HL-60 cells [21 ]. These observations correlate also with results from other groups who have reported an up-regulation of CCR5 after differentiation of primary monocytes from healthy donors [53 , 54 ]. It can be concluded that the Mono Mac 1 cell line is to date one of the only monocytoid cell lines showing properties similar to primary monocyte/macrophages in response to cellular differentiating agents.
Using single-round infection assays with recombinant
luciferase-encoding virions pseudotyped with T- and M-tropic Env
proteins, we were able to monitor permissiveness of both untreated and
treated Mono Mac 1 cells to viral infection. The final proof of the
susceptibility of Mono Mac 1 to virus infection was attained using
fully infectious T-, M-, and dual-tropic isolates of HIV-1. Viral
infection experiments revealed that T-tropic strains of HIV-1 poorly
replicate in both untreated and treated Mono Mac 1 cells. These results
do corroborate the study by Verani and co-workers who have reported
that primary human macrophages are highly resistant to infection with
T-tropic (CXCR4-dependent) TCLA strains of HIV-1 [34
].
However, untreated Mono Mac 1 were found to be highly susceptible to
infection by progeny viruses bearing M-tropic or dual-tropic envelope
proteins. Productive infection with M- and dual-tropic strains was
increased upon LPS-treatment of Mono Mac 1 cells. The observation that
LPS treatment leads to an increase in virus replication despite a
concomitant down-regulation of surface CD4 expression is not a surprise
considering that M-tropic isolates have already been shown to be less
dependent on the level of surface CD4 for infection to proceed
[55
]. Two different mechanisms, which are not mutually
exclusive, might be responsible for the observed higher virus
production in the LPS-treated Mono Mac 1 cells. The first scenario is
linked with the noticed enhancement of surface CCR5 expression (Fig. 1) and the second possibility is related with activation and nuclear
translocation of NF-B, a transcriptional factor known as a potent
activator of HIV-1 transcription [37
]. Measurement of
viral DNA by a semiquantitative PCR assay revealed that the enhancement
in virus production in LPS-treated Mono Mac 1 cells was not due to a
concomitant increase in the early steps in the virus life cycle, namely
the process of virus binding, fusion, entry, and reverse transcription.
The implication of NF-B was then studied based on the previously
reported activation of NF-B after LPS treatment of
monocyte/macrophages [56
57
58
59
]. Of interest to note is
the study by Frankenberger and colleagues showing that constitutive
NF-B in Mono Mac 1 cells can be further increased with LPS treatment
[40
]. Infectivity experiments conducted with
CCR5-independent virions made of the NL4-3 luciferase backbone
pseudotyped with A-MLV and VSV-G Envs allowed us to propose that
NF-B activation was most likely responsible for the increase in
virus production seen in LPS-treated Mono Mac 1. This idea was
confirmed by gel mobility shift assays, which revealed that higher
amounts of nuclear NF-B was detected in differentiated cells
(LPS-treated) compared with untreated Mono Mac 1 cells. Given that
NF-B strongly binds to the HIV-1 enhancer element, a region in the
viral regulatory sequences known to be essential for the induction of
HIV-1 transcriptional activity [42
43
44
], EMSAs were also
carried out using this sequence as a probe. Again, LPS treatment of
Mono Mac 1 cells was leading to an increase in NF-B binding activity
to the HIV-1 enhancer domain.
We also report here that the NSI isolate ADA (M-tropic) can form syncytia in LPS-treated Mono Mac 1 cells. Our data are consistent with previous observations indicating that Env proteins from M-tropic strains SF162, JR-FL, and BX08, which are also defined as NSI, can mediate cell-to-cell fusion [55 , 60 , 61 ]. Altogether, these findings indicate that the traditional nomenclature referring to the tropism of HIV-1 isolates as either NSI or SI [50 ] is no longer valid and should therefore be revised. It seems clear that the definition of HIV-1 tropism (i.e., T- vs. M-tropic) based on infection of established human cell lines does not adequately reflect the complex nature of virus-host cell interactions.
In summary, given that untreated Mono Mac 1 cells display properties of blood monocytes and that LPS treatment leads to the acquisition by Mono Mac 1 of a more mature phenotype (macrophage-like), this cell line can be used as a model system to study various aspects of monocytic functions. Mono Mac 1 is to date one of the few reported monocytoid cell lines that seems to effectively mimic primary human monocyte/macrophages in terms of surface antigen expression, cellular differentiation events and, as indicated by our findings, permissiveness to HIV-1 infection by M- and dual-tropic strains of HIV-1. The usefulness of this monocytoid cell line as a tool for studying several aspects of the life cycle of HIV-1 is clearly provided by the observation that differentiation of Mono Mac 1 results in an enhancement of virus replication. This finding is consistent with previous studies indicating that the permissiveness of primary human monocyte/macrophages for HIV-1 infection heavily depends on the state of cell differentiation [62 63 64 ]. Taken together, our data show that Mono Mac 1 represents a proper system to study, in the context of a human monocytoid malignant cell line, complex interactions occurring between HIV-1 and cells of the monocyte/macrophage lineage.
Received December 7, 1999; revised June 2, 2000; accepted July 10, 2000.
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, MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1 Science 272,1955-1959[Abstract]B) that actives transcription from a human immunodeficiency virus type 1 promoter in vitro Proc. Natl. Acad. Sci. USA 85,4700-4704B Nature 339,70-73[Medline]B responsive elements for initiation and tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes EMBO J 14,1552-1560[Medline]This article has been cited by other articles:
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  E. Cassol, M. Alfano, P. Biswas, and G. Poli Monocyte-derived macrophages and myeloid cell lines as targets of HIV-1 replication and persistence J. Leukoc. Biol., November 1, 2006; 80(5): 1018 - 1030. [Abstract] [Full Text] [PDF]
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