Human Cytomegalovirus Inhibits Cytokine-Induced Macrophage Differentiation

Abstract

Human cytomegalovirus (HCMV) infection in immunocompromised patients is associated with impaired immunological function. Blood
monocytes, which differentiate into macrophage effector cells, are of central importance for immune reactivity. Here, we demonstrate
that HCMV transiently blocks cytokine-induced differentiation of monocytes into functionally active phagocytic macrophages.
In HCMV-treated cultures, the cells had classical macrophage markers but lacked the classical morphological appearance of
macrophages and had impairments in migration and phagocytosis. Even at very low multiplicities of infection, macrophage differentiation
was almost completely inhibited. The inhibition appeared to be mediated by a soluble factor released upon viral treatment
of monocytes. Human immunodeficiency virus or measles virus had no such effects. These findings suggest that HCMV impairs
immune function by blocking certain aspects of cytokine-induced differentiation of monocytes and demonstrate an efficient
pathway for this virus to evade immune recognition that may have clinical implications for the generalized immunosuppression
often observed in HCMV-infected patients.

Full text

10.1128/JVI.78.19.10378-10389.2004.
2004, 78(19):10378. DOI:J. Virol.
Söderberg-Nauclér
Sara Gredmark, Tamara Tilburgs and Cecilia
Differentiation
Cytokine-Induced Macrophage
Human Cytomegalovirus Inhibits
http://jvi.asm.org/content/78/19/10378
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JOURNAL OF VIROLOGY, Oct. 2004, p. 10378–10389 Vol. 78, No. 19
0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.19.10378–10389.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Human Cytomegalovirus Inhibits Cytokine-Induced
Macrophage Differentiation
Sara Gredmark, Tamara Tilburgs, and Cecilia So¨derberg-Naucle´r*
Karolinska Systems Biomedicine Center, Department of Medicine, Center for
Molecular Medicine, Karolinska Institute, Stockholm, Sweden
Received 20 August 2003/Accepted 24 May 2004
Human cytomegalovirus (HCMV) infection in immunocompromised patients is associated with impaired
immunological function. Blood monocytes, which differentiate into macrophage effector cells, are of central
importance for immune reactivity. Here, we demonstrate that HCMV transiently blocks cytokine-induced
differentiation of monocytes into functionally active phagocytic macrophages. In HCMV-treated cultures, the
cells had classical macrophage markers but lacked the classical morphological appearance of macrophages
and had impairments in migration and phagocytosis. Even at very low multiplicities of infection, macrophage
differentiation was almost completely inhibited. The inhibition appeared to be mediated by a soluble factor
released upon viral treatment of monocytes. Human immunodeficiency virus or measles virus had no such
effects. These findings suggest that HCMV impairs immune function by blocking certain aspects of cytokine-
induced differentiation of monocytes and demonstrate an efficient pathway for this virus to evade immune
recognition that may have clinical implications for the generalized immunosuppression often observed in
HCMV-infected patients.
Human cytomegalovirus (HCMV), a member of the herpes-
virus family, is an opportunistic pathogen that causes serious
health problems in transplant recipients and in AIDS patients
(5). It is associated with atherosclerosis, restenosis after coro-
nary angioplasty, chronic rejection in organ transplant patients,
and chronic graft-versus-host disease in bone marrow trans-
plant recipients (15, 28, 51). After a primary infection, HCMV
persists in a latent form, and an estimated 60 to 100% of the
population carries the virus. In the latent phase, the viral ge-
nome exists in an episomal circular form (4) and does not
replicate, enabling the virus to avoid immune recognition. In-
creasing evidence suggests that HCMV infection adversely af-
fects both innate and adaptive immune responses. For exam-
ple, HCMV reduces the expression of HLA class I and II
molecules; inhibits antigenic processing, peptide presenta-
tion, and T-cell activation; confers resistance against natural
killer cells; and interferes with the humoral immune re-
sponse and cytokine-signaling events (reviewed in reference
26).
Neutrophils are considered the major cell type carrying the
virus during acute infection (11). However, monocytes are
thought to be responsible for dissemination of the virus and
are the predominant cell type harboring HCMV in the periph-
eral blood of seropositive individuals (54, 55). In CD14-posi-
tive monocytes, the HCMV genome is maintained at a rela-
tively low copy number (6 to 13 copies/cell) (46). HCMV
infection of monocytes is nonpermissive and restricted to early
events of gene expression (9). The absence of late gene expres-
sion and virus production are consistent with the hypothesis
that monocytes are reservoirs for latent virus. Although the
virus cannot replicate in monocytes, HCMV-infected macro-
phages expressing late viral genes have been identified in tissue
specimens from HCMV-infected patients (12).
Differentiation of monocytes into macrophages is a prereq-
uisite for productive HCMV infection (10, 18, 50, 56). It has
been shown that latent HCMV can be reactivated in differen-
tiated macrophages produced by allogeneic stimulation of pe-
ripheral blood mononuclear cells (PBMCs) (49). It has also
been shown that the inflammatory cytokines gamma interferon
(IFN-␥) and tumor necrosis factor alpha (TNF-␣) produced by
allogeneically stimulated T cells are important for the reacti-
vation of latent virus and the growth of HCMV in differenti-
ated macrophages (48). In myeloid cells, TNF-␣activates the
HCMV immediate-early (IE) promoter (37, 38, 40, 53). These
results suggest that immune activation and production of in-
flammatory cytokines are important for viral reactivation and
replication in HCMV-infected patients.
Since peripheral blood monocytes have a short half-life, the
reactivation of HCMV in macrophages suggests that HCMV
is maintained in a precursor population of the myeloid cell
lineage. Such cells would provide an ideal latency site for a
virus whose activation is closely linked to the immune system.
HCMV can infect CD34
⫹
pluripotent stem cells both in vitro
and in vivo (22, 29–31, 45, 62). CD33
⫹
granulocyte-macro-
phage progenitor cells derived from fetal liver can also be
infected with HCMV, and their differentiation into CD14
⫹
macrophages results in virus production (21, 23). However,
differentiation of all infected cells after viral entry would elim-
inate the virus during the infectious cycle.
We hypothesized that the establishment of latency by
HCMV in myeloid lineage cells affects cellular differentiation
signals. To test this hypothesis, we examined the effects of
HCMV on macrophage differentiation by assessing cell mor-
phology, expression of cell-surface markers, phagocytic activ-
ity, and cellular migration. Our results suggest that HCMV
* Corresponding author. Mailing address: Karolinska Institute, De-
partment of Medicine, Center for Molecular Medicine, L8:03, Karo-
linska Hospital, S-171 76 Stockholm, Sweden. Phone: 46-8-51779896.
Fax: 46-8-313147. E-mail: cecilia.soderberg.naucler@cmm.ki.se.
10378
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blocks the differentiation of monocytes into functionally active
macrophages, which may provide an efficient way for HCMV
to avoid immune recognition.
MATERIALS AND METHODS
Establishment of macrophage cultures. PBMCs from healthy donors (47)
were plated (Primaria Falcon dishes; Becton Dickinson, San Jose, Calif.) at 10 ⫻
10
6
to 18 ⫻10
6
cells/ml in Iscove’s modified medium with 2 mM L-glutamine, 100
U of penicillin/ml, 100 ␮g of streptomycin (Gibco BRL, Grand Island, N.Y.)/ml,
and 10% AB serum. After incubation at 37°C for 2 h, nonadherent cells were
removed, and the cultures were extensively washed and treated with supernatant
containing cytokines produced by an allogeneic reaction between T cells and
monocytes (allo-cytokines) to induce macrophage differentiation (50). Briefly,
PBMCs from different donors were mixed and incubated for 24 h in Iscove’s
complete medium. The supernatant was collected, cleared by centrifugation, and
used to stimulate separate monocyte cultures. In some experiments, cells were
restimulated once for 24 h at 1, 2, 6, and 18 days after treatment.
Separate monocyte cultures were also stimulated with recombinant IFN-␥(500
U/ml) and TNF-␣(10 ng/ml) (R&D Systems, Minneapolis, Minn.) either alone,
in combination, or with lipopolysaccharide (LPS) (1 ␮g/ml; Sigma) or with
phorbol myristate acetate (PMA) (10 ng/ml). Twenty-four hours after stimula-
tion, the cells were washed with Iscove’s medium and cultured in complete 60/30
medium (60% AIM-V medium, 30% Iscove’s modified medium, 10% AB serum,
L-glutamine, penicillin, and streptomycin). The medium was replaced with fresh
complete 60/30 medium every 3 to 4 days.
HCMV treatment. Monocyte-enriched cultures were washed with phosphate-
buffered saline (PBS) and then mock infected or infected with HCMV at a
multiplicity of infection (MOI) of 1 to 10. The cultures were stimulated at the
time of infection and incubated for 24 h at 37°C. Three HCMV strains were used:
AD169, Towne, and a clinical isolate (PO). Some cultures were infected with
human immunodeficiency virus (HIV) (viral titer, 1/10 or 1/1) or measles virus
(viral titer, 1/40 or 1/25); approximately 100% of monocytes are infected after
treatment with virus at these titers (Annika Lindhe and Anders So¨nnerborg
[Karolinska Institutet], personal communication).
Experiments were also performed with strain AD169 or Towne that had been
UV irradiated four times (Auto Cross Link, UV Stratalinker 1800; Stratagene)
or neutralized by incubation with intravenous immune globulin (IVIG) (50
mg/ml; Immuno AG, Vienna, Austria) for1hatroom temperature. Additional
experiments were performed with virus stocks that had been filtered through an
Acrodisc (0.1-␮m-pore-size filter; Gelman Sciences, Ann Arbor, Mich.) to re-
move 100% of the viral infectivity and with virus stock supernatant that had been
ultracentrifuged at 10,000 rpm for 16 h. The efficiency of these respective treat-
ments was tested by inoculating the respective preparations with human lung
fibroblasts, which showed that these treatments reduced infectivity by ⱖ99%.
Experiments were also performed with virus particles that had been pelleted at
10,000 rpm for 16 h and dissolved in 50 mM Tris HCl, 50 mM NaCl, and 10 mM
MgCl
2
at an MOI of approximately 1. Cell-free virus stocks were prepared from
supernatants of infected human lung fibroblasts, frozen, and stored at ⫺70°C.
Viral titers were determined by plaque assays (60).
In some experiments, supernatants from stimulated, HCMV-treated, or mock-
treated monocytic cultures were collected at 6, 12, or 24 h; cleared by centrifu-
gation; and stored at ⫺70°C until use. These supernatants were filtered to
remove virus particles, diluted 1:10 and 1:100, and used for infection or mock
infection of cultures.
Twenty-four hours after infection or mock infection, the cultures were washed
with Iscove’s medium and cultured in complete 60/30 medium. On day 7, the cells
were fixed, stained, and analyzed as described below.
Treatment with recombinant proteins and antibodies. At the time of stimu-
lation, the mock-treated cultures were also treated with recombinant IFN-␣(0.05
to 5 ng/ml) or recombinant IFN-␤(0.05 to 5 ng/ml), or the HCMV-treated
cultures were treated with antibodies to either IFN-␣(10 to 1,000 U) or IFN-␤
(10 to 2,500 U) (all from Biosource International, Camarillo, Calif.). The mock-
treated cultures were also treated with recombinant interleukin-6 (IL-6) (100 to
1,000 ng/ml) or recombinant IL-10 (1 ␮g/ml), and the HCMV-treated cultures
were treated with antibodies to either IL-6 and/or IL-6R (2.5 ␮g/ml) or with
antibodies to either IL-10 (10 ␮g/ml) and/or IL-10R (10 ␮g/ml) (all from R&D
Systems).
Measurement of cytokine production. Cytokine levels in the supernatant of
mock- and HCMV-treated cultures were measured with colorimetric sandwich
enzyme-linked immunosorbent assays (ELISAs) (for IFN-␣and IFN-␤, colori-
metric sandwich ELISA; PBL Biomedical Laboratories, Piscataway, N.J.; for
IL-6, IL-10, IL-12, and IL-1␤, Quantikine human colorimetric sandwich ELISAs;
BD Biosciences, San Jose, Calif.).
Quantification of macrophages. Differentiated macrophages are readily dis-
tinguished from monocytic cells by size, morphology, and the presence of nu-
merous cytoplasmic vacuoles (41). All monocytic cells except histiocytes and
specialized macrophages lack ␣-naphthyl acetate esterase enzyme activity. On
day 7 after mock treatment or HCMV treatment, cultures were fixed, stained
with an ␣-naphthyl acetate esterase staining kit (Sigma, St. Louis, Mo.), and
examined with an inverted microscope. The number of macrophages was deter-
mined by counting esterase-positive cells that exhibited a classical macrophage
appearance (histological phenotype).
At least 100 cells were counted in each well, and the percentage of monocytes
was calculated. Approximately 20% of cells in the monocyte-enriched cultures
(both HCMV-treated and mock-treated cultures) are not of monocytic origin,
but these numbers remain constant in different cultures from the same donor.
Hence, small cells can account for approximately 20% of the total cell number
(e.g., 20% of monocytes in mock-treated cultures represents complete differen-
tiation of most monocytic cells in the cultures). All experiments were performed
in duplicate with cells from at least 3 (generally 10) donors. Data are presented
as means ⫾the standard error of the mean (SEM). Statistical significance was
determined by paired, two-tailed ttests.
Flow cytometric analysis. The expression of cellular markers was assessed with
afluorescence-activated cell sorter (FACSort; Becton Dickinson). Adherent
macrophages were harvested by being scraped after a 60-min preincubation in
versene (Gibco BRL) at room temperature. The cells were stained with anti-
bodies recognizing different HLA class II molecules (HLA DR, DQ, and DP;
Becton Dickinson); HLA class I molecules (Dakopatts, Glostrup, Denmark); the
cell surface markers CD13 and CD14 (Dakopatts), CD40, CD68, and CD86; the
intracellular adhesion molecule (ICAM); or isotype controls (immunoglobulin
G1 and G2a) (Dakopatts). This was followed by treatment with appropriate
fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Dakopatts).
Expression levels were measured as mean channel fluorescence values compared
to the isotype control. The difference in the histogram mean channel value for
uninfected and HCMV-treated cells was calculated on a linear scale; a difference
of more than 10 channels between uninfected and HCMV-treated cells was
considered a positive or negative change, based on variations of controls.
Detection of HCMV replication. Seven days after infection, RNA from mock-
treated and HCMV-treated macrophages was prepared by adding Trizol (Gibco
BRL) to the cell cultures, and RNA was purified as previously described (47).
cDNA was synthesized with a first-strand cDNA kit (Pharmacia LKB Biotech-
nology, Uppsala, Sweden) and used as a template for HCMV-specific primer
pairs for the major early and pp150 genes in nested reverse transcription-PCR
assays. DNA was prepared as previously described (47). As a positive control for
the detection of DNA or RNA, primers specific for the glucose-6-phosphate
dehydrogenase gene were used for each sample (47). DNA and cDNA samples
from uninfected and HCMV-infected human lung fibroblasts were included as
positive and negative controls. The PCR products were visualized on 2% agarose
gels.
Phagocytosis assay. Seven days after HCMV treatment, yeast particles labeled
with FITC (42) were added to the cultures (420,000 particles/well in 200 ␮lof
60/30 medium) and incubated at 37°C. In some experiments, restimulation of
HCMV-treated cells was performed 48 h after initial infection, and the cultures
from mock, HCMV, and HCMV-restimulated cells were used after a total of 9
days in culture. As a negative control, cytochalasin D (1 ␮g/ml; Sigma) was added
to the cultures 30 min before the yeast particles. After 10, 30, or 60 min, phago-
cytosis was arrested by moving the plates to 4°C. The medium was replaced with
PBS containing 5% trypan blue to quench fluorescence from the extracellular
bound particles. Phagocytosis was quantified by fluorescence microscopy. Cells
that had ingested at least one yeast particle were counted after 10 and 60 min
(42). At least 50 to 100 cells/well were assessed in six independent experiments;
each experiment was performed in duplicate.
Migration assay. Chemotaxis assays were performed with 48-well polycarbon-
ate transwell culture chambers with 8-␮m pores (Costar, Cambridge, Mass.) (27).
Briefly, 100,000 cells in 100 ␮l of Iscove’s medium containing 10% AB serum, 2
mM L-glutamine, 100 U of penicillin/ml, and 100 ␮g of streptomycin (Gibco
BRL)/ml were added to the upper compartment, and 600 ␮l of serum-free
medium with either 100 ng of RANTES/ml, 100 ng of macrophage inflammatory
protein 1␣(MIP-1␣)/ml, or 50 ng of monocyte chemoattractant protein 1 (MCP-
1)/ml (all from R&D Systems) was added to the lower compartment. The cells
were incubated for 24 h at 37°C. The filters were washed three times in PBS, fixed
in methanol for 5 min, and washed once with PBS. The wells were stained with
Mayer hematoxylin and eosin (Histolab Products, Gothenburg, Sweden). The
cells from the upper side of the filter were removed with a cotton swab, and the
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cells on the lower side of each filter were counted in four representative fields by
light microscopy at 40⫻magnification.
RESULTS
HCMV inhibits cytokine-induced macrophage differentia-
tion. HCMV inhibited macrophage differentiation induced by
allo-cytokines in a dose-dependent fashion (Fig. 1A and B). At
an MOI of 1, all three HCMV strains completely inhibited
differentiation (Fig. 1C). HCMV also inhibited macrophage
differentiation induced by recombinant IFN-␥and TNF-␣or
by PMA or LPS (Fig. 1D). HCMV-treated monocytes were not
apoptotic (not shown).
AB
0
20
40
60
80
100
1 0.5 0.25 0.05 0.03 0.01 0
Viral Titer (MOI)
% Monocytes
0
10
20
30
40
50
60
70
80
90
100
AD169 Towne PO
% Monocytes
CD
Differentiated
Macrophages
HCMV Treated Cells Unstimulated
Monocytes
0
10
20
30
40
50
60
70
80
90
100
LPS
LPS HCMV
PMA
PMA HCMV
IFN/TNF
IFN/TNF HCMV
% Monocytes
FIG. 1. HCMV inhibits the differentiation of monocytes into macrophages. (A) Dose-dependent effects of infection with AD169 (MOI, 1 to
0.01) on macrophage differentiation. Values shown are means ⫾SEM for six separate experiments performed in duplicate. Esterase-negative cells
were subtracted for the data presentation. (B) Phase-contrast microscopy photograph of mock-treated, HCMV-treated (AD169), and unstimulated
monocytes at 7 days. Original magnification, ⫻40. (C) Effects of HCMV infection with AD169, Towne, or the clinical isolate PO at an MOI of
1. Values are means ⫾SEM for 10 separate experiments performed in duplicate. (D) Differentiation of monocytes stimulated with LPS, PMA,
or IFN-␥plus TNF-␣in the presence or absence of HCMV (AD169). Values are means ⫾SEM for three separate experiments performed in
duplicate. The differentiation was defined by esterase staining as well as by morphology.
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As shown by immunohistochemical staining, the expression
levels of CD40, CD68, HLA class II molecules, and the ICAM
were higher on differentiated macrophages than on monocytes
but were not affected by HCMV. In HCMV-treated cultures,
the macrophage markers CD13, CD64, CD68, CD86, and
ICAM-1, as well as HLA class I and class II molecules, were
expressed at high levels (Fig. 2A). CD14 levels, however, were
significantly lower in macrophages than in HCMV-treated or
freshly isolated monocytes (Fig. 2B and C), but varied between
donors. Therefore, the striking differences in morphology of
the cells after HCMV treatment were not accompanied by phe-
notypic differences, indicating that most, but not all, overt
aspects of monocyte differentiation were altered by the virus.
HCMV transiently inhibits differentiation. Next, we as-
sessed the time course of inhibition by treating monocytes with
HCMV at different times after stimulation with allo-cytokines.
HCMV treatment at 12 h inhibited macrophage differentiation
almost completely, but treatment at 48 or 72 h had no effect
(Fig. 3A), suggesting that the virus blocks early steps in differ-
entiation. We then assessed the effects of restimulating the cul-
tures with allo-cytokines for 24 h at different times after HCMV
treatment. In the absence of restimulation, no evidence of dif-
CD86
CD64
CD68
CD40
CD14
180
0
20
40
60
80
100
120
140
160
Monocytes
Macrophages
HCMV-treated
monocytes
***
HLA II
Mean Channel Expression
CD13
HLA I
ICAM
A
B
0
20
40
60
80
100
120
Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
Mean Channel Expression
Monocytes
Macrophages
HCMV-treated
monocytes
C
HCMV
Mock
FIG. 2. HCMV-treated monocytes retain high levels of CD14 expression. (A) Expression of cellular markers by uninfected monocytes,
macrophages, and HCMV-treated monocytes after 7 days in culture as determined by flow cytometric analysis. The data represent mean channel
fluorescence values ⫾SEM for five separate experiments. ***, P⬍0.001 (two-tailed, paired ttest; fresh monocytes versus HCMV-treated
monocytes). (B) CD14 expression of donors 1 to 5, comparing the mean channel fluorescence values for fresh monocytes, macrophages, and
HCMV-treated monocytes after 7 days in culture. (C) Representative histogram of CD14 expression by HCMV-treated monocytes and macro-
phages, assessed by flow cytometry.
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ferentiation was seen for up to 6 weeks. However, restimula-
tion 1 to 18 days after infection induced differentiation (Fig.
3B), which indicates that the viral inhibitory effect is transient.
HCMV-infected monocytes exhibit decreased phagocytosis
and migration. The differentiation of monocytes should be
associated with increases in macrophage functions such as cel-
lular migration in response to chemoattractants and phagocytic
activity. First, we tested the ability of mock-treated macro-
phages and HCMV-treated monocytes to migrate in response
to RANTES, MIP-1␣, and MCP-1 in a transwell filter system
7 days after infection. Migration of the HCMV-treated cells
at 24 h was reduced by 70 to 90% compared with migration
of mock-treated macrophages (Fig. 4A and B). Restimu-
lated HCMV treated cells were able to migrate in response to
RANTES to the same extent as mock-infected cells (Fig. 4C).
Similarly, phagocytosis of FITC-labeled yeast particles 7 days
after HCMV treatment or mock treatment was about 60%
lower in HCMV-treated monocytes than in mocked-treated
macrophages 60 min after addition of the yeast. Restimulation
of HCMV-treated cells resulted in phagocytosis at levels sim-
ilar to those of uninfected cells (Fig. 5). Cells pretreated with
cytochalasin D for 30 min did not phagocytose any yeast par-
ticles.
Differentiation is inhibited by a component of HCMV and
does not require viral replication. Since viruses other than
HCMV can impair immunological functions, we assessed the
effects of treatment with HIV or measles virus on cellular
differentiation. Neither virus inhibited macrophage differenti-
ation (Fig. 6A). Similarly, virus-free supernatants prepared by
filtration or ultracentrifugation of the medium from HCMV-
infected and mock-infected fibroblast cultures did not inhibit
differentiation. Concentrated virus particles, however, inhibit-
ed differentiation by 100% (Fig. 6B). Interestingly, pretreat-
ment of the virus with IVIG reduced infectivity by more than
99% (as tested on fibroblasts; data not shown) but did not affect
HCMV’s ability to block macrophage differentiation (Fig. 6C).
To determine if active HCMV replication was required to
block macrophage differentiation, monocytic cultures were
exposed to UV-inactivated, replication-deficient HCMV. The
cultures were not infected, but macrophage differentiation was
inhibited to levels similar to those obtained with untreated
virus (Fig. 6C).
A
0
20
40
60
80
100
120
0 3 6 12 24 48 72
Time after Stimulation (h)
% Monocytes
FIG. 3. HCMV’s ability to inhibit macrophage differentiation de-
clines with time after stimulation and is reversible. (A) Monocyte-
enriched cultures were stimulated with allo-cytokines; treated with
HCMV 0, 3, 6, 12, 24, 48, or 72 h after stimulation; and cultured for 7
days. Values are the means ⫾SEM for six separate experiments
performed in duplicate. Esterase-negative cells were subtracted for the
data presentation. (B) Monocyte-enriched cultures were stimulated
with allo-cytokines, treated with HCMV, and restimulated once for
24h1to18days after infection. Phase-contrast microscopy photo-
graphs are of mock-treated monocyte-derived macrophages, HCMV-
treated monocytes, HCMV-treated monocytes restimulated 6 days af-
ter infection, and HCMV-treated monocytes restimulated 18 days after
infection. The photographs were taken 7 days after restimulation.
Original magnification, ⫻40. The differentiation was defined by ester-
ase staining as well as by morphology.
B
HCMV + restim at 18 days
Mock-treated HCMV-treated HCMV + restim at 6 days
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Next, we analyzed RNA and DNA samples from mock-treated
and AD169-treated cells 1 and 6 weeks after infection. In a nested
PCR or reverse transcription-PCR assay with primers specific for
the HCMV major IE and pp150 genes, all HCMV-treated cells
were positive for HCMV DNA at both time points, but HCMV
RNA was not detected (data not shown), consistent with a latent
infection. Control samples from uninfected macrophage cultures
were negative for HCMV DNA and RNA (data not shown).
FIG. 4. HCMV-treated monocytes exhibit decreased migration in response to chemoattractants. (A) Cells were mock treated or treated with
HCMV and cultured for 7 days, and migration in response to RANTES, MIP-1␣, and MCP-1 was assessed with a transwell culture system. Cells
that migrated were counted by light microscopy in four representative fields at ⫻40 magnification. Values are the means ⫾SEM for three separate
experiments performed in duplicate. (B) Phase-contrast microscopy photographs of mock-treated macrophages or HCMV-treated monocytes that
migrated through the filter pores. Original magnification, ⫻40. Arrows are pointing at cells to exemplify migrated cells. (C) Cells were mock
treated, treated with HCMV, or restimulated 1 day postinfection after HCMV treatment. Migration in response to RANTES was assessed after
7 days in a transwell culture system. Values are the means ⫾SEM for three separate experiments performed in duplicate.
FIG. 5. HCMV-treated monocytes exhibit impaired phagocytosis. (A) After 7 days in culture, mock-treated and HCMV-treated monocytes
were tested for their ability to phagocytose FITC-labeled yeast particles. Cells that had phagocytosed yeast particles were quantified after 10 and
60 min. Values are means ⫾SEM for six separate experiments performed in duplicate. (B) Fluorescence microscopy photographs showing
phagocytosed yeast particles in mock-treated and HCMV-treated monocytes at 30 min. Original magnification, ⫻40. Arrows are pointing at cells
to exemplify phagocytosed FITC-labeled yeast particles. (C) Cells were mock treated, treated with HCMV, or restimulated 1 day postinfection
after HCMV treatment. Phagocytosis of FITC-labeled yeast particles was assessed after 7 days. Cells that had phagocytosed yeast particles were
quantified after 30 min. Values are the means ⫾SEM for three separate experiments performed in duplicate.
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Monocytes treated with HCMV release a soluble factor that
inhibits differentiation. HCMV infection can alter cytokine
production profiles, which could explain why monocytes chal-
lenged with HCMV failed to differentiate into macrophages.
To assess this possibility, we treated the cells with supernatants
collected at different times after HCMV treatment or mock
treatment. After filtration to remove virus particles, these su-
pernatants inhibited macrophage differentiation in a dose-de-
pendent manner, but those from mock-treated cultures did not
(Fig. 7A and B). These observations suggest that a soluble
factor(s) is released from monocytes upon HCMV treatment.
To identify such putative soluble factor that inhibited mac-
rophage differentiation, we tested the supernatants for IFN-␣,
IFN-␤, IL-1␤, IL-6, IL-10, and IL-12 by ELISAs. Only IFN-␣,
IL-6, and IL-10 were present at higher levels in supernatants
from HCMV-treated cultures than in those from mock-treated
cultures (Fig. 7C to E). Cytokine levels in the virus inocula
were very low or undetectable, and these results thus could not
explain the increased levels in the supernatants from HCMV-
treated cultures (data not shown).
We further assessed the individual effect on macrophage
differentiation of the different cytokines that were increased
upon HCMV treatment. While addition of increasing concen-
trations of recombinant IFN-␣and IFN-␤resulted in an inhi-
bition of macrophage differentiation, treatment of cells with
high doses of IL-6 did not (Fig. 8). High doses of recombinant
IL-10 inhibited macrophage differentiation by approximately
20% (Fig. 8B). Furthermore, we performed neutralization ex-
periments and found that antibodies against IFN-␣inhibited
the viral effect on macrophage differentiation by 25%, whereas
antibodies against IFN-␤or sheep serum did not affect HCMV’s
ability to inhibit macrophage differentiation (Fig. 8A). We were
not able to reverse the effect by HCMV on macrophage dif-
ferentiation by using antibodies specific for IL-10 and/or the
IL-10 receptor (Fig. 8B).
DISCUSSION
This study shows that HCMV blocks cytokine-induced mac-
rophage differentiation. HCMV-treated monocytes remained
small and morphologically distinct from differentiated macro-
phages and exhibited impairments in phagocytosis and migra-
tion. These findings are consistent with increasing evidence
that immune activation resulting in the production of inflam-
matory cytokines leads to macrophage differentiation and re-
activation of latent virus in HCMV-infected myeloid cells (2, 8,
25). Blocking the differentiation of monocytes into functionally
active macrophages may provide an efficient way for HCMV to
evade immune recognition.
The cell surface differentiation marker CD14 was expressed
at high levels by HCMV-treated cells, but its expression de-
creased during the differentiation of mock-treated cells. CD14
was thought to be a marker for myeloid lineage cells at differ-
ent stages of differentiation; however, its reactivity on mono-
cytes and macrophages varies depending on the specificity of
the antibodies used (20, 64). Early progenitor cells are CD14
negative; the majority of primary blood monocytes express high
levels of CD14, while alveolar macrophages are only weakly
immunopositive for CD14 (3, 35). Thus, our findings imply that
monocytic cells exposed to HCMV are arrested at a stage of
differentiation preceding maturation into macrophages.
The HCMV-induced inhibition of macrophage differentia-
tion appeared to be mediated by a component of the virus
particle and not by soluble factors produced by HCMV-in-
BC
0
20
40
60
80
100
120
Mock
HCMV
HIV 1/10
HIV 1/1
MV 1/25
MV 1/40
% Monocytes
A
0
20
40
60
80
100
120
Mock
HCMV
HCMV
UV
HCMV
IVIG
% Monocytes
0
20
40
60
80
100
120
Mock
HCMV
HCMV-
sup
UC-
HCMV
HL-sup
% Monocytes
FIG. 6. Inhibition of macrophage differentiation is not a general event after virus exposure and is mediated by a structural component of
HCMV. Macrophage differentiation after treatment of monocytic cultures with HIV or measles virus (MV) (A), ultracentrifuged virus-free
supernatants (HCMV-sup), concentrated virus particles (UC-HCMV), or supernatants from mock-infected cultures (HL-sup) (B), or HCMV
inactivated by treatment with UV or IVIG (HCMV UV and HCMV IVIG) (C). Values are means ⫾SEM for three separate experiments
performed in duplicate. The differentiation was defined by esterase staining as well as by morphology.
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fected fibroblasts in the virus stocks. Ultracentrifuged virus
inocula did not inhibit differentiation, whereas concentrated
HCMV particle preparations that had been cleared of soluble
factors and cytokines produced by the infected fibroblasts in-
hibited macrophage differentiation completely at an MOI of 1.
These observations suggest that the HCMV-induced inhibition
is directly dependent upon a protein component in the virus
particle.
UV inactivation did not prevent the virus from inhibiting
differentiation, indicating that viral replication was not re-
quired for the inhibitory effect. However, it is possible that
defective particles and UV-inactivated particles that cannot
produce infective virus particles support transcription from
specific areas of the viral genome. Alternatively, defective
HCMV particles might act as helper viruses and complement
replication, as suggested by higher titers of defective particles
than of infectious virus in HCMV preparations (57). Although
IVIG treatment reduced viral infectivity by 99%, it did not
affect the ability of the virus to inhibit macrophage differenti-
ation. Even at a neutralization rate of 99%, however, one
would expect to see at least a small reduction in the ability of
the virus to inhibit differentiation. That no reduction was seen
AB
0
10
20
30
40
50
60
70
80
90
100
% Monocytes
Mock
HCMV
6 h 12 h 24 hControl
0
10
20
30
40
50
60
70
80
90
100
Mock
HCMV
24 h Mock 1:10
24 h HCMV 1:10
24 h Mock 1:100
24 h HCMV 1:100
% Monocytes
CD
0
100
200
300
400
500
600
IFN-pg/ml
Mock
HCMV
6 h 12 h 24 h
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
IL-6 pg/ml
Mock
HCMV
6 h 12 h 24 h
0
50
100
150
200
250
300
IL-10 pg/ml
Mock
HCMV
24 h12 h6 h
E
FIG. 7. HCMV-treated monocytes release a soluble factor that blocks macrophage differentiation. (A) Effects of supernatants on macrophage
differentiation of monocytic cultures stimulated with allo-cytokines. Supernatants from HCMV-treated and mock-treated monocytic cultures were
collected 6, 12, and 24 h after HCMV treatment, cleared by centrifugation, filtered to remove infectious virus, and diluted 1:10. (B) Effects of
supernatants on macrophage differentiation of monocytic cultures stimulated with allo-cytokines. Supernatants from HCMV-treated and mock-
treated monocytic cultures were collected 24 h after HCMV treatment, cleared by centrifugation, filtered to remove infectious virus, and diluted
1:10 and 1:100. Values are means ⫾SEM for three separate experiments performed in duplicate. The differentiation was defined by esterase
staining as well as by morphology. (C to E) Levels of IFN-␣(undetectable IFN-␣levels in mock-treated cultures) (C), IL-6 (D), and IL-10 (E) as
detected by ELISA in the supernatants obtained from mock- and HCMV-infected cultures, respectively.
VOL. 78, 2004 HCMV INHIBITS MACROPHAGE DIFFERENTIATION 10385
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suggests that the inhibitory effect is mediated by nonimmuno-
genic components of the viral envelope or that determinants of
the viral glycoproteins involved in inducing inhibition of mac-
rophage differentiation were not blocked by antibodies in the
IVIG preparations.
HCMV treatment of monocytes inhibited differentiation
even at very low MOIs. This finding may be explained by the
fact that only a small percentage of the cells need to be ex-
posed to the responsible viral protein. Experiments with virus-
free supernatants obtained from monocytic cultures at differ-
ent times after HCMV treatment showed 50% inhibition of
differentiation by the 6-h supernatant and 80% inhibition by
the 12- and 24-h supernatants. Supernatants from mock-
treated cultures had no effect on differentiation. These results
FIG. 8. IFN-␣affects macrophage differentiation. (A) Recombinant IFN-␣and IFN-␤were added to monocytic cultures at the same time as
cytokine stimulation (IFN concentrations, 0.05 to 5 ng/ml). Antibodies against IFN-␣(10 to 1,000 U) and IFN-␤(10 to 2,500 U) and a control sheep
serum were added to the cultures at the same time point as HCMV treatment. (B) Recombinant IL-6 (100 to 1,000 ng/ml) or recombinant IL-10
(1,000 ng/ml) was added to the cultures at the same time as cytokine stimulation. Antibodies against IL-6 and/or IL-6 receptor or antibodies against
IL-10 and/or the IL-10-receptor were added at the same time point as HCMV treatment. The differentiation was defined by esterase staining as
well as by morphology.
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imply that HCMV treatment of monocytes leads to release of
a soluble factor that inhibits surrounding cells from differen-
tiating into macrophages.
Supernatants from the HCMV-treated monocytic cultures
contained higher levels of IL-6, IL-10, and IFN-␣, suggesting
that HCMV induces the secretion of one or more cytokines
that can inhibit differentiation. The most likely candidate is
IFN-␣, which was not present in the viral inoculum but was
detected at increasing levels in the supernatants from virus-
treated monocytes. In support of this hypothesis, PBMCs ex-
posed to HCMV in vitro produced IFN-␣within 4 to 10 h (34),
which largely accounted for the observed immunosuppressive
effects, including reduced morphological differentiation of
monocytes and decreased oxidative activity. We found here
that exogenously added IFN-␣and IFN-␤were both able to
inhibit the differentiation of macrophages. Recombinant IL-10
and a viral IL-10 homologue (24) inhibit the proliferation of
mitogen-stimulated PBMCs (52), and IL-10 prevents the dif-
ferentiation of monocytes into dendritic cells and instead pro-
motes their maturation into macrophages (1, 16). Similarly,
IL-6 upregulates the expression of functional receptors for
macrophage colony-stimulating factor on monocytes, which
alters their differentiation from a dendritic cell phenotype to a
macrophage phenotype (7). We found that high doses of re-
combinant IL-10, but not IL-6, were able to inhibit macro-
phage differentiation by approximately 20%. Interestingly, only
antibodies to IFN-␣, but not to IL-6 or IL-10, were able to
partly reverse the viral inhibition of macrophage differentia-
tion. However, we were not able to completely reverse the
inhibition of macrophage differentiation in this experimental
system; the neutralizing antibodies to IFN-␣only reversed
25% of the inhibitory effect, which suggests that multiple cy-
tokines may be involved in this process. Alternatively, released
IFN-␣may be rapidly internalized by the cells in the culture
and hence not affected by exogenous antibodies.
How does the virus benefit from blocking cytokine-induced
macrophage differentiation? One possibility is that blocking
enables the virus to avoid immune recognition in the early
phase of infection. Another is that HCMV infection in undif-
ferentiated myeloid cells results in latency, consistent with the
presence of HCMV DNA, but not RNA, in the cells 6 weeks
after exposure to HCMV, when the monocytes remained small
and did not exhibit the classical morphological appearance of
macrophages. HCMV did not induce apoptosis in the virus-
treated cells, and its inhibitory effect was reversible. After
several weeks in culture, the monocytes differentiated into
macrophages upon restimulation with allo-cytokines, and the
restimulated cells had the same ability to migrate and phago-
cytose as mock-treated macrophages. This is important be-
cause latently infected cells must undergo differentiation be-
fore latent virus can be reactivated. We found that HCMV
transiently blocks cytokine-induced differentiation of mono-
cytes into macrophages. Thus, latent virus might be activated
when monocytes encounter an inflammatory response, enter
tissues, and differentiate into macrophages.
Cellular differentiation is a prerequisite for HCMV replica-
tion in cells that normally undergo differentiation. For exam-
ple, HCMV infection in monocytes occurs only at a low fre-
quency, is abortive, or is restricted to expression of IE genes
(18, 44). In contrast, when monocytes differentiate into mac-
rophages, a high proportion of cells becomes productively in-
fected by HCMV (10, 18, 50). Cellular differentiation is also
important for productive HCMV infection of human terato-
carcinoma cells, which become permissive after differentiation
is induced by retinoic acid (13). Since both adsorption and
penetration of virus occur to the same extent in monocytes and
teratocarcinoma cells (13, 18), the inability of the virus to
replicate in undifferentiated cells is not caused by a virus entry
block. The virus may be inactive in monocytes early in the
infectious process, as a result of a direct inactivating effect of
HCMV on the infected cells. Hence, the virus-induced inhibi-
tion of cellular differentiation may facilitate the establishment
of latency. However, the viral and cellular mechanisms for
maintaining HCMV latency are largely unknown.
Transcripts associated with HCMV latency have been iden-
tified in HCMV-infected, fetal liver-derived CD33
⫹
granulo-
cyte-macrophage progenitor cells in vitro and in HCMV-sero-
positive individuals and may play a critical role in the control of
latency (17). These transcripts are represented by sense (open
reading frame 94 [ORF94]) and anti-sense (ORF152 and
ORF154) transcripts expressed from a region of the genome
(UL122-UL123) normally involved in the expression of the
transcriptional activators (IE1 and IE2) that participate in lytic
replication (23). Recently, Goodrum et al. developed a model
for HCMV latency in hematopoietic progenitor cells, in which
CD34
⫹
cells were infected with HCMV and cultured (14).
These cells did not produce infectious virus during the culture
period, but virus reactivation could be induced by coculture
with human fibroblasts. As shown by microarray analysis, the
patterns of viral gene expression were distinctly different dur-
ing latent, productive, and nonproductive infections (14).
In our study, HCMV-treated monocyte cultures examined 1
and 6 weeks after infection contained viral DNA but no viral
RNA, indicating that the cells were neither abortively nor
productively infected. We did not detect latency-associated
transcripts by our HCMV IE-specific primers, but we cannot
exclude the existence of gene expression from a latency-asso-
ciated region of the HCMV genome that could not be detected
with these primers. Recent observations also suggest that in-
activation of ORF94 does not affect reactivation of latent
HCMV in CD33
⫹
granulocyte-macrophage progenitor cells
derived from fetal liver (61). In addition, a previous study did
not detect these transcripts in allogeneically stimulated mac-
rophages (49). Hence, it is unclear whether these transcripts
are critical for maintaining latent virus in undifferentiated
monocytes. However, immune activation with concomitant
production of proinflammatory cytokines appears to be essen-
tial for the reactivation and replication of HCMV in infected
patients (2, 8, 25).
Our findings may have important clinical implications.
HCMV predisposes infected subjects to other opportunistic
infections and is associated with bacterial and fungal infections
in transplant patients (32, 36, 59), possibly by altering the
function of monocyte-derived macrophages and their progen-
itor cells during infection (6, 33, 39, 43, 58). HCMV also
impairs monocyte functions, for example, by suppressing the
production of IL-1 and IL-2 in PBMCs and by decreasing the
proliferative response of these cells to mitogens in vitro (19).
HCMV infection also inhibits monocyte differentiation by in-
ducing IFN-␣from nonadherent PBMCs (34). In our study,
VOL. 78, 2004 HCMV INHIBITS MACROPHAGE DIFFERENTIATION 10387
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HCMV-infected monocytes had a decreased ability to migrate
in response to chemokines and to phagocytose yeast particles.
As a result of their decreased ability to present peptides in the
context of major histocompatibility complex class II molecules,
HCMV-infected monocytes may be impaired in their ability to
clear other infections, resulting in immunosuppression.
Other viruses also cause immunosuppression. HIV induces
immunosuppression mainly by targeting CD4
⫹
T cells, and
measles virus both inhibits T-cell proliferation and induces
functional abnormalities in actively infected monocytes (33,
63). Cells infected with measles virus are also impaired in their
ability to present antigens, despite induced expression of major
histocompatibility complex molecules. In our study, neither
HIV nor measles virus inhibited macrophage differentiation,
which suggests that HCMV’s effect on cellular differentiation is
not a general event after viral exposure.
In conclusion, our findings provide new tools to examine
cellular differentiation response pathways and may help in the
development of new strategies to improve immunological func-
tions in HCMV-infected patients.
ACKNOWLEDGMENTS
We thank Mireille Vossen for technical assistance, Annika Linde for
measles virus, Anders So¨nnerborg for HIV and access to P3 facilities,
and Erna Mo¨ller, William B. Britt, Daniel Streblow, and Lena Ser-
rander for helpful discussions.
This work was supported by grants from the Swedish Medical Re-
search Council (K98-06X-12615-01A and K2001-16X-12615-04A to
C.S.-N. and K202-06X-00793-37C to E.M.), the Tobias Foundation
(1313/98, 20/01, and 33/02), the Swedish Children’s Cancer Research
Foundation (1998/065 and 01/046), the Heart-Lung Foundation
(199941305 and 200241138), the Swedish Society for Medicine (1999-
02-0347), the Goljes Foundation (325, 520), and the Emil and Wera
Cornells Foundation. C.S.-N. is a fellow of the Wenner-Gren Foun-
dation, Sweden.
ADDENDUM IN PROOF
Interestingly, Smith et al. recently published data which sug-
gest that HCMV instead could induce differentiation of mono-
cytes into macrophages (M. S. Smith, G. L. Bentz, J. S. Alex-
ander, and A. D. Yurochko, J. Virol., 78:4444–4453, 2004).
Collagen-coated plates were used in their experimental system,
which may explain the different results obtained, and, impor-
tantly, these differences may also reflect differences in the in
vivo situation in which monocytes in the peripheral blood or
monocytes entering tissues as macrophages will become
HCMV infected.
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