Monocyte heterogeneity underlying
phenotypic changes in monocytes
according to SIV disease stage
Woong-Ki Kim,*,1Yue Sun,* Hien Do,†Patrick Autissier,* Elkan F. Halpern,‡
Michael Piatak Jr.,§Jeffrey D. Lifson,§Tricia H. Burdo,??Michael S. McGrath,†,¶
and Kenneth Williams*,2
*Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, and‡Department of Radiology, Massachusetts General
Hospital, Harvard Medical School, Boston, Massachusetts, USA;†Pathologica, LLC, San Francisco, California, USA;§AIDS
Vaccine Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, USA;??Department of
Biology, Boston College, Chestnut Hill, Massachusetts, USA; and¶Department of Laboratory Medicine, Positive Health
Program, University of California, San Francisco, California, USA
RECEIVED FEBRUARY 16, 2009; REVISED AUGUST 28, 2009; ACCEPTED AUGUST 31, 2009. DOI: 10.1189/jlb.0209082
Infection by HIV is associated with the expansion of
monocytes expressing CD16 antigens, but the signifi-
cance of this in HIV pathogenesis is largely unknown. In
rhesus macaques, at least three subpopulations of blood
monocytes were identified based on their expression of
CD14 and CD16: CD14highCD16?, CD14highCD16low, and
CD14lowCD16high. The phenotypes and functions of these
subpopulations, including CD16?monocytes, were inves-
tigated in normal, uninfected rhesus macaques and ma-
caques that were infected with SIV or chimeric SHIV. To
assess whether these different monocyte subpopulations
expand or contract in AIDS pathogenesis, we conducted
a cross-sectional study of 54 SIV- or SHIV-infected ma-
caques and 48 uninfected controls. The absolute num-
bers of monocyte populations were examined in acutely
infected animals, chronically infected animals with no de-
tectable plasma virus RNA, chronically infected animals
with detectable plasma virus RNA, and animals that died
with AIDS. The absolute numbers of CD14highCD16lowand
CD14lowCD16highmonocytes were elevated signifi-
cantly in acutely infected animals and chronically in-
fected animals with detectable plasma virus RNA
compared with uninfected controls. Moreover, a sig-
nificant, positive correlation was evident between the
number of CD14highCD16lowor CD14lowCD16highmono-
cytes and plasma viral load in the infected cohort.
These data show the dynamic changes of blood
monocytes, most notably, CD14highCD16lowmono-
cytes during lentiviral infection, which are specific to
disease stage. J. Leukoc. Biol. : 000–000; 2010.
HIV infection in humans results in hematologic abnormalities
and immune suppression, which is well represented by
changes in the CD4/CD8 T cell ratio. As obviously seen in T
cell populations, HIV-induced immune perturbation is also
easily observed within the mononuclear phagocyte system in-
cluding macrophages and monocytes. Circulating monocytes
in HIV disease display marked phenotypic and functional alter-
ations associated with progression to AIDS [1–5]. Notably, cell
surface antigens that exhibit a bimodal expression on all of
the resting monocyte populations (thereby indicating the pres-
ence of subpopulations) have been recognized as the most
affected by HIV infection. For example, an increase in Fc?RIII
(CD16) expression and decrease in Fc?RI (CD64) and L-selec-
tin (CD62L) expression on blood monocytes were significant
in HIV-infected patients compared with uninfected controls
[6–12]. It is likely that these changes in HIV-infected patients
are largely a result of expansion and contraction of monocyte
subpopulations expressing these markers. Indeed, it has been
shown repeatedly that CD14?monocytes, expressing CD16,
expand during HIV infection [13–18] and SIV infection [19–
21]. We have observed recently that monocytes leave the bone
marrow and enter the blood in response to monocyte/macro-
phage apoptosis in monkeys that are SIV-infected .
The presence of phenotypically definable subpopulations
of human monocytes has been recognized for over 20 years.
1. Current address: Department of Microbiology and Molecular Cell Biology,
Eastern Virginia Medical School, Norfolk, VA 23507, USA.
2. Correspondence at current address: Department of Biology, Boston Col-
lege, Chestnut Hill, MA 02467, USA. E-mail: email@example.com
Abbreviations: APC?allophycocyanin, CCR2?C–C motif chemokine re-
ceptor 2, CD14highCD16?monocytes?monocytes with a high density of
CD14 and no CD16 antigen, CD14highCD16low?monocytes that coexpress
high levels of CD14 and low levels of CD16, CD14lowCD16highmonocytes?
monocytes that coexpress low levels of CD14 and high levels of CD16,
CD62L?CD62 ligand, CX3CR1?C–X3–C motif chemokine receptor 1,
Cy5.5?cyanin 5.5, DC?dendritic cell, HAART?highly active antiretroviral
therapy, mDC?myeloid dendritic cell, pDC?plasmacytoid dendritic cell,
The online version of this paper, found at www.jleukbio.org, includes
0741-5400/10/00-0001 © Society for Leukocyte Biology
Volume , MONTH 2010
Journal of Leukocyte Biology 1
Epub ahead of print October 20, 2009 - doi:10.1189/jlb.0209082
Copyright 2009 by The Society for Leukocyte Biology.
Using two-color flow cytometry of human monocytes for the
correlated expression of CD14 (LPS receptor) and CD16,
Ziegler-Heitbrock et al.  first showed two monocyte sub-
populations: CD14highCD16?and CD14lowCD16high. Follow-
ing this initial description, refined analyses of chemokine
receptors and adhesion molecules have better defined sub-
populations of monocytes and shown additional phenotypic
heterogeneity of human monocytes [24–27]. These data
show a CD14highCD16?subpopulation of monocytes that ex-
press CD62L, CD64, and CCR2 with low levels of CX3CR1
expression. In contrast, the CD14lowCD16highsubpopulation
expresses no detectable CD62L, CD64, or CCR2 but high
levels of CX3CR1.
Studies in mice have demonstrated in addition to their pheno-
typic differences differential homing potential of i.v.-injected
(two rodent counterparts of human CD14highCD16?and
CD14lowCD16highmonocytes, respectively) [28, 29]. A more
recent study by Auffray et al.  showed that mouse
CD11a?CX3CR1highCCR2?Gr1?monocytes (equivalent to
human CD14lowCD16highmonocytes) are the first to exit
into inflamed tissues. In this study, Auffray et al.  dem-
onstrated that this subset crawls on normal endothelium of
blood vessels, invades tissues rapidly upon tissue damage,
and becomes tissue macrophages. This study is also in line
with the work showing that the human counterpart
CD14lowCD16highmonocytes preferentially reside in the
marginal pool and are mobilized quickly .
Functional in vitro studies analyzing endocytotic and phago-
cytotic functions and the ability to produce inflammatory cyto-
kines in response to LPS also confirmed the existence of two
distinct monocyte subsets in humans [14, 32]. It is important
to note the presence of a third, less well-described subpopula-
tion that expresses high levels of CD14 and low levels of CD16
(CD14highCD16lowmonocytes). Adding one additional layer of
complexity to monocyte heterogeneity, these monocytes with
an “intermediate” or “transient” phenotype may also represent
a subset with distinctive functions. Their role in HIV and SIV
pathogenesis is understudied.
SIV- or chimeric SHIV-infected rhesus macaques are the prime
model to study HIV pathogenesis. In this study, we investigated
the phenotypic and functional differences of monocyte subpopu-
lations in normal, uninfected and SIV- or SHIV-infected monkeys.
We initially identified by flow cytometry four distinct subpopula-
tions of blood mononuclear phagocytes in rhesus monkeys based
on differential expression of CD14 and CD16: CD14highCD16?,
CD14highCD16low, CD14lowCD16high, and CD14?/lowCD16?. To
better characterize these subpopulations, we have further exam-
ined the expression of a broad spectrum of other monocyte- and
DC-associated markers. The differential expression of these mark-
ers was largely confirmed by gene array studies using FACS-sorted
monocyte subpopulations. To investigate the relative contribution
of subsets that replicate virus in vivo, we examined levels of SIV
RNA associated with freshly sorted monocyte subpopulations in
infected animals and found their differential ability to sustain
SIV infection. Changes in monocyte subpopulations during lenti-
virus infection were assessed by a cross-sectional study of the un-
infected and infected cohorts of rhesus macaques. In this report,
by performing a comprehensive analysis of the association be-
tween lentiviral infection and changes in monocytes, we identify
disease-specific changes in monocyte subpopulations.
MATERIALS AND METHODS
One hundred and two rhesus macaques (Macaca mulatta) were recruited
for this study, including 48 uninfected control macaques and 54 animals
infected with SIV or SHIV. The infected cohort of monkeys consisted of
animals infected with SIVmac239 (n?3), SIVmac251 (n?30), SHIV-89.6P
(n?14), and SHIV-SF162P3 (n?7), all of which have similar magnitudes of
peak viremic, chronic infection and AIDS-related plasma virus. Most blood
samples were collected once at necropsy, from killed animals that were not
allowed to develop AIDS and death or from animals that died with AIDS.
Four animals were used for serial blood samples, two to three times over
the course of disease.
Flow cytometric immunophenotyping
Peripheral blood samples were used to determine the immune phenotype
of rhesus monocytes. We and others [33, 34] found that heparinized blood
is not suitable to assess expression of some monocyte-associated antigens as
a result of high, nonspecific background staining (e.g., CCR2) or inconsis-
tency of staining (e.g., CD163). Similarly, as circulating immune complexes
bind CD16 that mask recognition by anti-CD16 antibodies , it is essen-
tial to remove plasma to detect CD16 consistently. Therefore, all subse-
quent immunofluorescence staining for flow cytometry was performed us-
ing EDTA-anticoagulated, plasma-depleted blood. For this, aliquots of 100
?L EDTA-anticoagulated whole blood were washed with PBS, and the pel-
lets were incubated at room temperature for 15 min with a mixture of anti-
bodies conjugated with FITC, PE, PerCP/PerCP-Cy5.5, or APC. Three four-
color immunophenotyping panels were used: One panel included anti-
CD14-FITC (clone M5E2, BD PharMingen, San Diego, CA, USA), anti-
CD16-PE (3G8, BD PharMingen), and anti-HLA-DR-PerCP (L243, Becton
Dickinson, San Diego, CA, USA); the second panel included anti-CD16-PE,
anti-HLA-DR-PerCP, and anti-CD14-APC; and the third panel included anti-
CD16-FITC, anti-CD14-PerCP-Cy5.5, and anti-HLA-DR-APC. Using these
three basic panels, monocyte subpopulations were examined further for
expression of other monocyte- and DC-associated antigens. The mAb used
for this included: CD1c-APC (AD5-8E7, Miltenyi Biotec, Auburn, CA, USA),
CD11b-APC (M1/126.96.36.199, Miltenyi Biotec), CD11c-APC (S-HCL-3, Bec-
ton Dickinson), CD31-APC (WM59, eBioscience, San Diego, CA, USA),
CD45RA-FITC (2H4, Beckman Coulter, Fullerton, CA, USA), CD45RB-
FITC (PD7/26, Dako, Denmark), CD62L-PE (SK11, BD PharMingen),
CD64-FITC (10.1, BD PharMingen), CD163-FITC (Mac2-158, Trillium Diag-
nostics, Brewer, ME, USA), CD123-PE (7G3, BD PharMingen), CD141-PE
(1A4, BD PharMingen), CCR2-APC (48607, R&D Systems, Minneapolis,
MN, USA), CCR8 (191704, R&D Systems), and CX3CR1-APC (rabbit poly-
clonal, Torrey Pines Biolabs, Houston, TX, USA). Isotype control mAb
were used in all panels. These included: mouse IgG2b-APC (27-35, BD
PharMingen), rat IgG2b-APC (141945, R&D Systems), mouse IgG1-APC
(MOPC-21, BD PharMingen), mouse IgG1-FITC (MOPC-21), mouse IgG2a-
FITC (G155-178, BD PharMingen), and mouse IgG2a-PE (G155-178). After
staining, cells were fixed in 2% paraformaldehyde and analyzed on a FACS-
Calibur (BD Biosciences, San Jose, CA, USA). To establish the flow cyto-
metric identification of monkey monocytes in blood, a gate was set based
on light-scatter properties of blood monocytes to contain predominantly
monocytes. The monocyte gate constituted 80–90% CD14?cells in all sam-
ples tested, and contaminating CD3?T cells were consistently found to be
?5%. Isotype controls were used to set gates for positive cells, allowing
?1% positive cells in the negative control samples. Absolute counts of
monocytes and monocyte subsets were calculated by multiplying the per-
centage of each subpopulation within the blood monocyte gate by the
number of monocytes/?L blood, as determined by complete blood cell
counts (ADVIA 120 hematology analyzer, Bayer Diagnostics, Germany).
Journal of Leukocyte Biology
Volume , MONTH 2010
Lymphocyte immunophenotyping was performed using combinations of
following mAb: CD3-PerCP-Cy5.5 or -APC (SP34, BD PharMingen); CD4-
FITC (19thy5d7, Beckman Coulter) or CD8-FITC or -PE (SK1, Becton Dick-
inson); and CD20-PerCP-Cy5.5 (L27, Becton Dickinson), CD28-PE (28.2,
Beckman Coulter), and CD95-APC (DX2, BD PharMingen). Naı ¨ve T lym-
phocytes were determined by their CD28?CD95?phenotype in CD3?CD4?
or CD3?CD8?cells, whereas central and effector memory T cell subsets
were defined as being CD28?CD95?and CD28?CD95?, respectively.
Isolation of monocyte subpopulations
FACS sorting of monocyte subpopulations was performed as described pre-
viously . Briefly, Ficoll-separated PBMC isolated from monkey blood
were stained with anti-CD14-FITC, anti-CD16-PE, anti-CD4-PerCP-Cy5.5, and
anti-CD3-APC. Cell sorting was done using a BD FACSVantage flow cytome-
ter in a Biosafety Level 2? facility. CD3-negative cells in the monocyte gate
were fractionated based on CD14 and CD16 expression into CD14high
CD16?, CD14highCD16low, and CD14lowCD16highcells. Sorted cells were
washed and snap-frozen rapidly for gene array or SIV-DNA and -RNA analy-
sis described below. In addition, CD3?CD4?lymphocytes were sorted for
comparison. Purity of all FACS-sorted populations was confirmed by flow
cytometry and was routinely ?95%.
Intracellular cytokine staining
The phenotype and frequency of nonstimulated monocytes spontaneously
producing TNF-? and granzyme B in normal, uninfected and SIV-infected
animals were determined by intracellular cytokine staining followed by sev-
en-color flow cytometry. PBMC were isolated from heparinized blood by
Ficoll gradient centrifugation and then incubated at 37°C in a 5% CO2in-
cubator for 3 h in the presence of monensin (GolgiStop, BD Biosciences).
Cultured cells were stained with mAb specific for cell surface molecules,
including anti-CD3-Pacific Blue (SP34-2, Becton Dickinson), anti-CD4-Am-
Cyan (L200, Becton Dickinson), anti-CD8-PerCP-Cy5.5 (RPA-T8, Becton
Dickinson), anti-CD16-PE (3G8, BD PharMingen), and anti-CD14-PE-Texas
Red (RMO52, Beckman Coulter). Cells were fixed and permeabilized with
Cytofix/Cytoperm solution (BD Biosciences) and stained with anti-TNF-?-
FITC (mAb11, BD PharMingen) and anti-granzyme B-Alexa Fluor 700
(GB11, BD PharMingen). Labeled cells were resuspended and fixed with
1% formaldehyde in PBS. Samples were collected and analyzed on a BD
LSR II instrument using FlowJo software (Tree Star, Ashland, OR, USA).
Events (500,000–1 million) were collected/sample.
Determination of plasma viral RNA and
cell-associated viral RNA and DNA
Copies of plasma SIV and SHIV RNA and cell-associated SIV RNA and
DNA were determined using a quantitative real-time RT-PCR assay as de-
scribed previously .
The Kruskal-Wallis test was used for nonparametric-independent group
comparisons of the medians. Only if the test were significant (P?0.05)
were comparisons of each of the infected groups with an uninfected con-
trol group performed using the two-tailed Wilcoxon test. All combinations
of the independent variables were analyzed with 95% confidence level for
each variable. Spearman correlation coefficients were computed between
two of all variables.
Phenotypic heterogeneity of monkey mononuclear
phagocytes in blood
Within the blood, the mononuclear phagocyte system is repre-
sented by monocytes, which can be identified by flow cytom-
etry as cells within a “gate” of high forward-scatter and inter-
mediate side-scatter. This light-scatter gate contained cells dis-
playing different levels of CD14 expression (Fig. 1A). Flow
cytometric analysis of cells within the gate for the correlated
expression of CD14 and CD16 delineated four distinct sub-
populations. A major subpopulation is CD14highCD16?. Two
additional subpopulations express variable levels of CD14 and
CD16: CD14highCD16lowand CD14lowCD16high. A fourth popu-
lation has CD14low/?CD16?. This CD14low/?CD16?population
is heterogeneous, containing immature monocytes,
CD1cCD11bCD11cCD31 CD45RA CD64CD68
CD86 CD163 CCR2CX3CR1 HLA-DR MAC387TLR2
Forward ScatterCD14-APC CD14-APC
CD14− −CD16− −
Figure 1. Phenotypic heterogeneity of blood mononuclear phagocytes
in monkeys. (A) Definition of mononuclear phagocytes in peripheral
blood of rhesus macaques. (Left) Typical light-scatter contour map of
whole blood cells; Ly, lymphocytes; Mo, monocytes; G, granulocytes
(middle) overlay of gated mononuclear phagocytes (Mo; green) on
side-scatter versus CD14 contour profile (red); (right) correlated ex-
pression of CD14 and CD16 defines four distinct subpopulations of
mononuclear phagocytes in normal monkey blood. The regions indi-
cated in the CD14 versus CD16 contour plot of gated mononuclear
phagocytes were used to differentiate the following monocyte and DC
populations in the blood. (B) Phenotypic analysis of blood monocyte
subpopulations. The expression of indicated molecules was deter-
mined by four-color flow cytometry. The histograms exhibit the phe-
notypic characteristics of the three monocyte subpopulations under
study: CD14highCD16?, CD14highCD16low, and CD14lowCD16high. The
mean fluorescence intensity is shown. The discriminators shown were
set at the foot of the isotype control. The results for each marker are
representative of at least 10 uninfected animals. (C) Phenotypic char-
acterization of CD14low/?CD16?cells (CD14–CD16–). These cells are
heterogeneous, containing CD11c?mDCs and CD123?pDCs (left
panel). CD11c?mDCs express another DC marker CD1c (middle).
Some CD14low/?CD16?cells express CD40 antigen, unlike monocyte
Kim et al.
Monocyte heterogeneity in SIV infection
Volume , MONTH 2010
Journal of Leukocyte Biology 3
CD1c?CD11c?mDCs, and CD1c?CD123?pDCs, as well as
lymphocytes (Fig. 1C). Monocyte subpopulations,
CD14highCD16?, CD14highCD16low, and CD14lowCD16high, in
normal, uninfected macaques (n?48) comprised 65% ? 8%
(mean?sd), 8% ? 4%, and 9% ? 4%, respectively, of the to-
tal monocytes, gated on forward- versus side-scatter.
To better define the cells of these subpopulations, we ex-
amined their expression of monocyte- and DC-associated
markers including CD1c, CD11b, CD11c, CD31, CD40,
CD45RA, CD64, CD68, CD86, CD123, CD163, CCR2,
CX3CR1, HLA-DR, MAC387, and TLR2 (Fig. 1B). CD1c
(blood DC antigen-1) expression in the CD14highCD16?
population was bimodal and demonstrated CD1c?or CD1c?
phenotypes. Virtually, all of the CD14highCD16lowcells were
CD1c?, whereas all CD14lowCD16highcells were CD1c?. CD11b
expression was uniformly high on CD14highCD16?and
CD14highCD16lowcells but low on CD14lowCD16highcells. CD11c
expression was bimodal in the CD14highCD16lowpopulation with
CD11c?and CD11c?subsets. Essentially, all of the CD14low
CD16highcells were CD11c?, and all of the CD14highCD16?cells
were CD11c?. PECAM-1 (CD31) was expressed on nearly all cells
in the CD14highCD16?, CD14highCD16low, and CD14lowCD16high
populations, with the highest levels on CD14lowCD16highcells.
CD45RA expression was limited to a subset of the CD14low
CD16highpopulation. All CD14highCD16?cells and a majority of
CD14highCD16lowcells were CD64?, but no cells expressing this
marker were detected in the CD14lowCD16highpopulation. A sub-
set of CD86-positive cells was detected in all of the populations,
where the highest levels of CD86 were found on CD14high
CD16lowcells. CD163, a macrophage marker, was expressed on
all CD14highCD16?and CD14highCD16lowcells but not on
CD14lowCD16highcells. The expression of CCR2 followed the pat-
tern of CD64. High CX3CR1 was found on all cells in CD14high
CD16lowand CD14lowCD16high, whereas CX3CR1lowcells were
detected in about one-half of the CD14highCD16?population;
the level of CX3CR1 expression on CD14highCD16lowcells was
greater than that on CD14lowCD16highcells. Virtually, all of the
CD14highCD16?, CD14highCD16low, and CD14lowCD16highcells
were HLA-DR?, and CD14highCD16lowcells had the highest lev-
els. Overall, these data demonstrate that the CD14highCD16?,
CD14highCD16low, and CD14lowCD16highpopulations comprise
the majority of blood monocytes/macrophages, and the
CD14low/?CD16?cells are heterogeneous and consist of mDC
and pDC, CD3?T lymphocytes, and likely, monocyte precursors.
Table 1 summarizes the data described above with regard to the
CD14highCD16?, CD14highCD16low, and CD14lowCD16highpheno-
It has been suggested that within blood, especially during
disease states, populations of monocytes are present with phe-
notypes similar to maturing or mature macrophages. We ex-
amined the different monocyte populations for two such mark-
ers: intracellular expression of CD68 (a pan-macrophage
marker) and MAC387 (a heterodimer of S100A8 and S100A9,
expressed by monocytes, which is down-regulated and lost dur-
ing macrophage differentiation) antigens. We found two mu-
tually exclusive subsets: a CD68?MAC387?subset and
CD68?MAC387?subset. All CD14highCD16?and CD14high
CD16lowcells were CD68?MAC387?, and a majority
of CD14lowCD16highcells were CD68?MAC387?, suggesting
that compared with CD14highCD16?and CD14highCD16low
cells, the CD14lowCD16highpopulations represent more mature
and macrophage-like cells.
To investigate whether CD16?monocytes are comprised of
distinct subpopulations of monocytes rather than a continuum of
CD14?monocytes with differing levels of cell activation, we used
gene array analysis that compared overall gene expression pro-
files of FACS-sorted CD14highCD16?, CD14highCD16low, and
CD14lowCD16highcells (Supplemental Data Section). Microarray
analysis confirmed the phenotypic differences between these sub-
populations, differentiated on the basis of markers described
above (Supplemental Fig. 1) and discerned further between these
subpopulations by revealing additional differences (Supplemental
Tables). In comparison with CD14highCD16?cells, a large num-
ber of genes (9098/29361, 30.9%) were expressed differentially
in CD14highCD16lowand CD14lowCD16highcells, underscoring
the fundamental differences between CD16?and CD16?mono-
cytes (Supplemental Table 1). Thirty-one genes and 94 genes
were associated specifically with CD14highCD16lowand
CD14lowCD16highsubpopulations (Supplemental Tables 2 and 3).
A relatively small set of genes that was expressed differentially
between the two CD16?subpopulations highlights similarity be-
tween the two cell types, but differentially expressed genes of
function observed in CD14highCD16lowand CD14lowCD16high
subpopulations suggest different roles that these two subpopula-
tions may play in vivo.
Blood monocyte subpopulation dynamics during SIV
In an effort to understand how the distribution of monocytes
and monocyte subpopulations changes in HIV/SIV infection,
we analyzed SIV- or SHIV-infected (n?54) and naı ¨ve (n?48)
TABLE 1. Monkey Monocyte Subpopulations: Phenotypic
Characteristics of Three Identified Subpopulations
Determined by Flow Cytometry Are Summarized
CD14 and CD16 were listed first, as these markers were used to differ-
entiate the monocyte populations.
Journal of Leukocyte Biology
Volume , MONTH 2010
rhesus monkeys (Table 2). The percentage of total monocytes
was significantly higher in infected animals than in uninfected
controls (mean?sd; infected, 7.5%?5.3% vs. uninfected,
4.5%?1.7%; P?0.01, unpaired t-test). Additionally, the expan-
sion of monocytes in blood, termed monocytosis (defined as
?500 cells/?L), was seen more frequently in infected animals
(20/54, 37.4%) than in uninfected animals (6/48, 12.5%).
However, the mean absolute numbers of total monocytes were
not significantly different between the infected and uninfected
groups (506?582 and 327?170 counts/?L, respectively). The
percentage and absolute number of monocytes in the infected
cohort were highly variable and displayed a more positively
skewed distribution as compared with uninfected controls
(skewness of the absolute numbers, 5.58 and 1.06, for the in-
fected vs. uninfected animals). For these reasons, we analyzed
the data from these cohorts using nonparametric tests of me-
To characterize in detail distribution patterns of monocyte
subpopulations reflecting the progression of the disease, we
stratified infected monkeys using the following disease stages
at the time of specimen collection: (1) acute phase (2 weeks
p.i.; n?6) and chronic phase (?12 weeks p.i.); (2) without
(n?16) or (3) with (n?26) detectable plasma virus RNA; and
(4) terminal AIDS (at necropsy; n?6; Table 2A). The absolute
numbers of total monocytes were significantly higher in chron-
ically infected monkeys with detectable plasma virus RNA (me-
dian 370 counts/?L, P?0.0392, Wilcoxon rank-sum test) and
monkeys with terminal AIDS (median 685 counts/?L, P?
0.0083, Wilcoxon rank-sum test) than in normal, uninfected
controls (median 285 counts/?L). In acutely infected animals,
the monocyte number (median 265 counts/?L) was compara-
ble with that in uninfected controls, and the median monocyte
percentage was significantly higher than in uninfected controls
(P?0.0014, Wilcoxon rank-sum test). In acutely infected ani-
mals, monocytes expressed lower levels of CD14 (data not
shown), in confirmation of previous reports [19, 36]. The me-
dian absolute numbers of both CD16?monocyte subpopula-
tions (CD14highCD16low: 66 counts/?L; and CD14lowCD16high:
47 counts/?L) were significantly greater in acutely infected
animals than in uninfected controls (CD14highCD16low:
21 counts/?L; and CD14lowCD16high: 24 counts/?L; Table
2A). In parallel to these increases, the relative percentage of
the CD14highCD16?population decreased significantly
(P?0.0003, Wilcoxon rank-sum test). This may indicate that a
rapid shift in the circulating monocyte pool occurred 2 weeks
p.i. from the CD14highCD16?subpopulation to
Whether this occurs in blood or from cells emerging from
bone marrow is not clear.
The distribution of monocyte subpopulations in chronically
infected animals with no detectable plasma virus is comparable
with that in uninfected controls (Table 2A). In contrast, in
chronically infected animals with detectable plasma virus,
CD14highCD16lowand CD14lowCD16highsubpopulations in-
creased significantly (medians 53 and 41 counts/?L, respec-
tively), and the absolute number of the CD14highCD16?sub-
population (median 194 counts/?L) was similar to that in un-
infected controls (median 172 counts/?L). This indicates that
TABLE 2A. Distribution of Monocyte Subpopulations
CD4 T cells
Uninfected control (n?48)
Peak viremic (n?6)
Infected with no detectable
plasma virus RNA (n?16)
Infected with detectable plasma
virus RNA (n?26)
Terminal AIDS (n?6)
P values were determined using the Wilcoxon rank-sum test for nonparametric data and refer to comparisons of each animal group of infected animals with uninfected control ani-
mals. Significance indicated withaP ? 0.001;bP ? 0.01;cP ? 0.05;dP ? 0.0001. M2/M1, CD14highCD16low-to-CD14highCD16–; M3/M1, CD14lowCD16high-to-CD14highCD16–; (M2?M3)/
M1, (CD14highCD16low?CD14lowCD16high)-to-CD14highCD16–; NA, not applicable; ND, not determined.
Kim et al.
Monocyte heterogeneity in SIV infection
Volume , MONTH 2010
Journal of Leukocyte Biology 5
CD14highCD16lowand CD14lowCD16highsubpopulations were
expanded during this stage and that the marginal rise in the
total monocyte count could be exclusively a result of the ex-
pansion of CD14highCD16lowand CD14lowCD16highsubpopula-
tions. This chronically infected group had highly variable
plasma viral load among animals, and these variations may re-
sult in variations in the distribution of monocyte subpopula-
tions. Therefore, chronically infected animals with detectable
plasma virus were stratified further into two potential sub-
groups, according to median viral load (70,000 copies/mL):
those with viral load ?70,000 copies/ml (the low viral load
subgroup, n?14) and those with viral load ?70,000 copies/ml
(the high viral load subgroup, n?12; Table 2B). Absolute
numbers of total monocytes and CD14highCD16lowand
CD14lowCD16highsubpopulations were significantly and mark-
edly higher in the high viral load subgroup than in the low
viral load subgroup. In fact, in the low viral load subgroup, the
distribution of monocyte subpopulations is similar to that in
the uninfected cohort.
The animals that died with AIDS had significantly greater
numbers of monocytes than uninfected controls. Absolute
numbers of the CD14highCD16?, CD14highCD16low, and
CD14lowCD16highpopulations were found increased markedly
in this group as compared with the uninfected cohort. In the
animals that died with AIDS, the median number of the
CD14highCD16lowsubpopulation was highest among all of the
groups, and the difference with the uninfected controls almost
achieved statistical significance (P?0.0592, Wilcoxon rank-sum
test). The median number of the CD14lowCD16highpopulation
in this group was also highest, but this increase was not statisti-
cally significant (P?0.1965). Two animals with terminal AIDS
had strikingly low counts of CD14lowCD16highcells (five and
six counts/?L), whereas the other four animals had high
CD14lowCD16highcell numbers of 279, 150, 106, and 95
To minimize interindividual variations in the numbers of
monocyte subpopulations, the ratio of CD14highCD16low,
CD14lowCD16high, and both to CD14highCD16?in individual
monkeys was calculated. The median CD14highCD16low/
CD14highCD16?ratio in uninfected controls was 0.12. In the
acutely infected animals, this ratio increased and was changed
significantly in favor of the CD14highCD16lowpopulation (1.02,
P?0.0002). The CD14highCD16low/CD14highCD16?ratio in
chronically infected animals with no detectable plasma virus
(0.19) or with low viral load (0.18) is comparable with that of
uninfected controls. Then, the ratio increased to median levels
of 0.38 in the high viral load subgroup and 0.36 in the AIDS
group. The CD14lowCD16high/CD14highCD16?ratio followed
the similar pattern, but an increase of this ratio in the AIDS
group was not significant. Collectively, these data indicate that
the distribution of monocyte subpopulations was altered in a
manner specific to disease stages, where an expansion of cells
with more mature macrophage-like properties emerges.
Immunologic correlations with monocyte
For each animal, important immunological parameters of HIV
infection, including CD4?and CD8?T-lymphocyte counts, the
TABLE 2B. Distribution of Monocyte Subpopulations
CD4 T cells
n?14 Monkeys with VL
n?12 Monkeys with VL
Animals with detectable plasma virus RNA are subdivided into two subgroups: one with low viral load (?median, 70,000 copies/ml); and one with high viral load (?70,000 copies/ml).
Comparison of animals with high viral load vs. animals with low viral load performed with the Wilcoxon test. Significance at **, P ? 0.01; ***, P ? 0.001, and ****, P ? 0.0001.
Journal of Leukocyte Biology
Volume , MONTH 2010
number of CD4?and CD8?central memory T cells, and
plasma viral load, were analyzed in parallel to monocyte sub-
sets. We sought to determine in this cross-sectional study
whether any immunologic parameter correlates with the
changes in distribution of monocyte subpopulations. Initially,
we found significant correlations between some of these pa-
rameters and monocyte subpopulations, especially the
CD14highCD16low, when we used a pool of all of the animals
studied. Upon further investigation, we realized that these cor-
relations were mostly attributed to disease-specific clustering of
data. Thus, correlations were sought separately in the unin-
fected and infected cohorts (Table 3). In the uninfected con-
trols, the CD4 and CD8 T cell counts were positively corre-
lated with numbers of total monocytes and CD16?monocytes
(CD14highCD16lowand CD14lowCD16high). However, we found
significant negative correlations between the CD4/CD8 ratio and
monocyte subpopulations. Interestingly, CD8?central memory T
cells were negatively correlated with monocytes and monocyte
subpopulations with strong Spearman correlation coefficients,
suggesting a role of CD8?central memory T cells in the control
of the numbers of monocytes. We have found recently similar
observations in HIV-infected humans . In infected animals,
viral load is the only parameter amongst those tested in our study
that correlated with monocytes and monocyte subpopulations.
Statistically significant, positive correlations were found between
the CD14highCD16lowcount and the viral load (r?0.3757,
P?0.0085) or between the CD14lowCD16highcount and the viral
load (r?0.3781, P?0.0081), suggesting that virus or viral proteins
could play a role in controlling monocyte dynamics.
Functional heterogeneity of monocyte subpopulations
in SIV infection
The phenotypic and transcriptional heterogeneity of monocyte
subpopulations and their dynamics in SIV infection suggest
that there exist functionally distinct subsets. We examined the
expression of TNF and the replication of SIV in these subsets.
Differential ex vivo expression of TNF in monocyte subsets.
CD14?monocytes/macrophages are the dominant source of
TNF-? in vivo, and it has been shown that the CD14lowCD16high
monocyte is a major source of TNF in response to LPS in vitro.
To determine which subsets express this cytokine, intracellular
cytokine staining was used to compare the phenotype and fre-
quency of monocytes spontaneously producing (without in
vitro stimulation) TNF-? as well as granzyme B in monkeys
with subacute (n?5; 8 weeks p.i.) and chronic (n?8; 2.6 years
p.i.) SIV infection and uninfected animals as controls (n?5).
Monocytes from normal, uninfected controls expressed little-
to-no TNF-? but produced granzyme B at low frequency.
Monocytes from infected monkeys produced TNF-? spontane-
ously, where expression of TNF was preferentially associated
with CD14highCD16?and CD14highCD16lowcells but not with
the CD14lowCD16high(Fig. 2). The percentage of TNF-express-
ing cells in the CD14highCD16?population was 7.5–25 times
greater than that in the CD14lowCD16high. The frequency of
monocyte subsets spontaneously producing TNF-? was in-
creased significantly in infected monkeys compared with unin-
fected controls. The magnitude of the increase in frequency
was greater in chronically infected animals than in subacutely
infected animals. These results support SIV infection-depen-
dent, spontaneous production of TNF-?, which may reflect the
immune activation in infected monkeys. Granzyme B was ex-
pressed exclusively in CD14lowCD16highcells, and the fre-
quency of granzyme B-expressing cells in the CD14lowCD16high
population in infected animals was not significantly higher
than that in uninfected controls (infected, mean?sd,
17.25%?13.38% vs. uninfected, mean?sd, 9.43%?7.43%). It
appears that granzyme B expression is not associated specifi-
cally with SIV infection.
Differential susceptibility to SIV infection in monocyte sub-
sets. Peripheral blood monocytes are a major reservoir for
HIV-1 in infected individuals on virally suppressive HAART
[38, 39]. We examined levels of SIV-DNA and -RNA associated
with monocyte subpopulations in acutely infected animals (12
days p.i., n?4; Fig. 3). Although levels of SIV DNA were com-
parable in all monocyte subpopulations (data not shown;
P?0.05), significantly higher levels of SIV RNA were found in
TABLE 3. Correlations Between Immunologic Parameters and Monocyte Subpopulations
Animal group Monocyte count
Uninfected control (n?48)
CD4 T cells
CD8 T cells
CD4 CM T cells
CD8 CM T cells
CD4 T cells
CD8 T cells
CD4 CM T cells
CD8 CM T cells
Plasma viral loadc
–0.7199 (0.0001) –0.5994 (0.0025) –0.5617 (0.0053) –0.5874 (0.0032)
0.4089 (0.0039) 0.3757 (0.0085)0.3781 (0.0081)
aThe correlation coefficients were calculated using the Spearman’s rank test.bP values are shown in parentheses. The nonsignificant correlation
coefficients are not presented.cn ? 48. CM, Central memory.
Kim et al.
Monocyte heterogeneity in SIV infection
Volume , MONTH 2010
Journal of Leukocyte Biology 7
CD14highCD16lowcells (P?0.05, paired t-test). However, the
level of viral replication in CD14highCD16lowcells was not com-
parable with that of CD4?T lymphocytes. SIV replication,
which occurred preferentially in CD14highCD16lowcells, likely
involves high levels of cell-specific factors in CD14highCD16low
cells such as SerpinB2 (Supplemental Table 2).
The present study was designed to identify monkey monocyte
subpopulations by gene array and flow cytometry and to char-
acterize the distribution of these subpopulations during SIV
infection. Our data demonstrate for the first time large-scale
gene expression differences between CD16?monocytes
(CD14highCD16?) and the two CD16?subpopulations
(CD14highCD16lowand CD14lowCD16high). Between the two
CD16?subpopulations, differential expression of relatively few
but distinct genes was found (Supplemental Data Section).
Expansion of these CD16?monocytes during SIV infection
was associated with SIV disease stages and correlated to plasma
viral load, suggesting effects of virus infection on monocyte
Immature monocytes are derived from CD34?myeloid pro-
genitor cells and become mature in the bone marrow. Mono-
cytic maturation takes place in sequential stages, which is con-
trolled by sequential expression of a specific set of transcrip-
tion factors including PU.1, acute myelogenous leukemia 1,
C/EBP?, IFN regulatory factor-8, and MafB [40–42]. The se-
quential expression and combination of these transcription
factors lead to gradual loss of CD34 phenotype and sequential
acquisition of CD11a, CD33, CD11b, CD14, and CD16 pheno-
types [43, 44]. Mature CD14?monocytes continuously leave
bone marrow, enter the circulation, and marginate to the ves-
sel wall. The monocytes that pass through the vessel wall be-
come tissue macrophages.
This transit population, however, appears not to be homoge-
neous. It has been suggested that bone marrow monocytes are
kinetically heterogeneous in their transit time through bone mar-
row [45, 46]. Blood monocytes are heterogeneous in terms of
physical and functional properties. Two or more subsets of mono-
cytes that differ in size, density, or granularity [47–49] have been
isolated, and they show differential peroxidase and lysozyme pro-
duction, cytokine production, phagocytotic and cytotoxic activi-
ties, and responses to GM-CSF [50–53]. Phenotypically, the heter-
ogeneity of human blood monocytes can be demonstrated by the
presence of CD16?monocytes. As CD16 is expressed only on a
minor subset of mature CD14highmonocytes in bone marrow
[44, 54], CD14?monocytes that express CD16 in bone marrow
and peripheral blood represent a subpopulation at the more ma-
ture differentiation stages. Our genome-wide transcriptional pro-
filing demonstrates the transcriptional heterogeneity of monkey
monocytes and identifies CD16?monocytes as even more mature
cell types than mature CD16?monocytes (CD14highCD16?). Our
study differentiated CD16?monocytes further into two subpopu-
% TNF-α α+cells
CD14highCD16low CD14lowCD16highCD14− −CD16− −
Figure 2. Differential expression of
TNF-? and granzyme B in mono-
cyte subpopulations. (A) The ex-
pression of TNF-? and granzyme B
in monocyte subpopulations. Intra-
cellular expression of TNF-? and
granzyme B was assessed with intra-
cellular cytokine staining, followed
by flow cytometry. CD3?, CD4?, or
CD8?cells were excluded from the
CD14?monocyte population to
ensure no contaminating cells. The
figure shown was the analysis of
one chronically infected animal
with a detectable viral load; repre-
sentative of n ? 8. (B) The expres-
sion of TNF-? in peripheral blood
monocyte subsets defined by CD14
and CD16 was evaluated in healthy,
uninfected monkeys (n?5), mon-
keys with subacute SIV infection
(n?5), and monkeys with chronic
SIV infection (n?8). Results are
expressed as means ? sd; signifi-
cant at *, P ? 0.05, and **, P ?
0.01 (one-way ANOVA followed by
Journal of Leukocyte Biology
Volume , MONTH 2010
lations: CD14highCD16lowand CD14lowCD16high. Although
CD14highCD16lowcells appeared phenotypically intermediate be-
tween CD14highCD16?and CD14lowCD16highin terms of expres-
sion levels of CD11c, CD31, CD64, and CCR2, CD14highCD16low
cells do not seem to represent a transient subpopulation from
the CD14highCD16?to the CD14lowCD16high. The highest levels
of CD1c, CD86, CX3CR1, HLA-DR, and TLR2 were expressed on
CD14highCD16lowcells. Our gene array data showed that the
gene expression profile of the CD14highCD16lowpopulation is
substantially different from that of the CD14highCD16?and
closely shared with that of the CD14lowCD16high(Supplemental
Data Section). In addition, we showed that CD14highCD16lowcells
are a functionally distinct subpopulation that preferentially takes
up dextran polymers, produces TNF, and supports SIV replica-
tion. Recently, it was shown that influenza virus infection and
severe asthma are associated with this particular subpopulation
[55–57]. Our study also reports for the first time a “cytotoxic”
phenotype (granzyme?lymphotoxinß?perforin?TRAIL?), which
specifically defines CX3CR1highCD14lowCD16highcells. Whether
CD14highCD16lowcells are the precursor of CD14lowCD16high
cells or whether CD14highCD16lowand CD14lowCD16highcells are
macrophage-like cells with two different phenotypes remains to
Monocytes are susceptible targets for HIV infection, and
macrophages, their tissue counterparts, constitute an impor-
tant reservoir of virus, especially during the chronic phase of
infection. A study of the relationship between monocyte differ-
entiation and HIV replication demonstrated that restricted
HIV replication and no evident cytopathic effects in mono-
cytes are important mechanisms of virus persistence . In-
deed, monocytes function as a major reservoir for persisting
virus during antiretroviral therapy . In a cohort of 39 HIV-
positive patients, increases in the number of HIV-positive
monocytes in the blood preceded the increase in the plasma
viral load, suggesting their contribution to viral load . On
the other hand, HIV infection appears to induce phenotypic
and functional changes in monocytes. Myelomonoblastic cells,
upon in vitro HIV infection, become a more mature, mono-
cytic phenotype . In HIV-infected patients, phenotypic and
functional changes driven by HIV infection occurred in the
monocyte population, which includes expansion of CD16?
monocytes [8, 13, 16, 61, 62]. It is important to note that only
a small fraction of the monocyte populations becomes infected
and that infection by HIV is not needed to develop such dys-
functions. In vitro HIV gp120 stimulation is sufficient to in-
duce dysfunctions and phenotypic changes in monocytes/mac-
rophages [63–65]. HIV gp120 is known to induce IL-10 pro-
duction in monocytes [66, 67] and induce the ability of
monocytes to kill CD4?T cells , which may contribute to
HIV-induced immune suppression in vivo. Notably, gp120 mim-
ics the expansion of CD16?monocytes, which is observed in
the blood of HIV-infected patients, increasing CD16 expres-
sion on cultured monocytes . We suggest that HIV infec-
tion induces differential monocyte maturation and kinetics,
thereby affecting the heterogeneity of monocytes in the pe-
Relevance of this heterogeneity in the pathogenesis of HIV
is beginning to be understood. It has been shown recently that
in HIV-infected patients receiving HAART, CD16?monocytes
are more susceptible to HIV infection and harbor preferen-
tially HIV DNA . A direct comparison between this human
study and our monkey study cannot be made, as Ellery et al.
 measured HIV DNA detected in sorted CD16?monocytes
including CD16lowand CD16highsubsets, whereas we exam-
ined separately SIV RNA detected in CD16?monocytes, which
were fractionated further into CD14highCD16lowand CD14low
CD16highmonocytes. We found that SIV RNA is detected in all
three monocyte subsets with a highest level in CD14high
CD16lowmonocytes. Our data in SIV-infected monkeys are
somewhat different than these human data. One reason for
this might be species differences, where SIV is detected readily
in monocytes, and HIV is less abundant in human monocytes
[20, 21, 38, 39, 69, 70]. Another difference is that Ellery et al.
 analyzed HIV DNA from patients on HAART, and we as-
sessed SIV RNA from animals that were not on antiretroviral
therapy. The relationship between phenotypes and functions
of different monocyte subpopulations remains to be estab-
lished. Interestingly, a recent mouse study by Auffray et al.
 demonstrated CD11a?CX3CR1highGr1?monocytes
(equivalent to human CD14lowCD16highmonocytes) as a subset
that crawls on normal endothelium of blood vessels, invades
tissues rapidly upon tissue damage, and becomes tissue macro-
phages. Whether such cells do so in the course of viral infec-
tion has yet to be studied thoroughly.
We were interested in establishing the contribution of differ-
ent monocyte subpopulations in HIV pathogenesis. In this
study, we have conducted a cross-sectional study of SIV- or
SHIV-infected animals to investigate changes in the distribu-
tion of monocyte subpopulations during infection. We report
the positive correlations between plasma viral load and abso-
lute numbers of total monocytes and the two CD16?subpopu-
SIV RNA Copy Eq./105 cells
Figure 3. Differential levels of SIV replication in monocyte subpopula-
tions, which with CD4?T cells, were FACS-sorted from the blood of
acutely infected animals (12 days p.i., n?4). Levels of SIV RNA associ-
ated with each cell type were measured by quantitative RT-PCR; signifi-
cant at *, P ? 0.05, and **, P ? 0.01 (paired t-tests).
Kim et al.
Monocyte heterogeneity in SIV infection
Volume , MONTH 2010
Journal of Leukocyte Biology 9
lations. Expansion of CD16?monocytes was evident in the
acute and chronic phase of infection with high plasma viral
load, suggesting a biphasic increase (first phase early after pri-
mary infection and second phase prior to development of
AIDS) in the absolute number of CD16?monocytes. Expan-
sion or a depletion of CD16?monocytes was seen in cases of
terminal AIDS. This may signify impaired maturation at the
late chronic state of the disease. Collectively, our observations
suggest that monocyte maturation and/or activation are re-
lated to plasma viral load.
This work was supported by Public Health Service grants
NS040237 (K. W.), NS037654 (K. W.), NS0500041 (K. W.),
and MH81835 (K. W. and M. S. M.).
1. Bender, B. S., Davidson, B. L., Kline, R., Brown, C., Quinn, T. C. (1988)
Role of the mononuclear phagocyte system in the immunopathogenesis
of human immunodeficiency virus infection and the acquired immunode-
ficiency syndrome. Rev. Infect. Dis. 10, 1142–1154.
2. Braun, D. P., Kessler, H., Falk, L., Paul, D., Harris, J. E., Blaauw, B., Lan-
day, A. (1988) Monocyte functional studies in asymptomatic, human im-
munodeficiency disease virus (HIV)-infected individuals. J. Clin. Immunol.
3. Rich, E. A., Toossi, Z., Fujiwara, H., Hanigosky, R., Lederman, M. M., Ell-
ner, J. J. (1988) Defective accessory function of monocytes in human im-
munodeficiency virus-related disease syndromes. J. Lab. Clin. Med. 112,
4. Dudhane, A., Conti, B., Orlikowsky, T., Wang, Z. Q., Mangla, N., Gupta,
A., Wormser, G. P., Hoffmann, M. K. (1996) Monocytes in HIV type 1-in-
fected individuals lose expression of costimulatory B7 molecules and ac-
quire cytotoxic activity. AIDS Res. Hum. Retroviruses 12, 885–892.
5. Noursadeghi, M., Katz, D. R., Miller, R. F. (2006) HIV-1 infection of
mononuclear phagocytic cells: the case for bacterial innate immune defi-
ciency in AIDS. Lancet Infect. Dis. 6, 794–804.
6. Allen, J. B., Wong, H. L., Guyre, P. M., Simon, G. L., Wahl, S. M. (1991)
Association of circulating receptor Fc ? RIII-positive monocytes in AIDS
patients with elevated levels of transforming growth factor-?. J. Clin. In-
vest. 87, 1773–1779.
7. Locher, C., Vanham, G., Kestens, L., Kruger, M., Ceuppens, J. L., Vinger-
hoets, J., Gigase, P. (1994) Expression patterns of Fc ? receptors,
HLA-DR and selected adhesion molecules on monocytes from normal
and HIV-infected individuals. Clin. Exp. Immunol. 98, 115–122.
8. Trial, J., Birdsall, H. H., Hallum, J. A., Crane, M. L., Rodriguez-Barradas,
M. C., de Jong, A. L., Krishnan, B., Lacke, C. E., Figdor, C. G., Rossen,
R. D. (1995) Phenotypic and functional changes in peripheral blood
monocytes during progression of human immunodeficiency virus infec-
tion. Effects of soluble immune complexes, cytokines, subcellular particu-
lates from apoptotic cells, and HIV-1-encoded proteins on monocytes
phagocytic function, oxidative burst, transendothelial migration, and cell
surface phenotype. J. Clin. Invest. 95, 1690–1701.
9. Hayes, P. J., Miao, Y. M., Gotch, F. M., Gazzard, B. G. (1999) Alterations
in blood leucocyte adhesion molecule profiles in HIV-1 infection. Clin.
Exp. Immunol. 117, 331–334.
10. Pillet, S., Prevost, M. H., Preira, A., Girard, P. M., Rogine, N., Hakim, J.,
Gougerot-Pocidalo, M. A., Elbim, C. (1999) Monocyte expression of ad-
hesion molecules in HIV-infected patients: variations according to disease
stage and possible pathogenic role. Lab. Invest. 79, 815–822.
11. Miller, L. S., Atabai, K., Nowakowski, M., Chan, A., Bluth, M. H., Minkoff,
H., Durkin, H. G. (2001) Increased expression of CD23 (Fc(?) receptor
II) by peripheral blood monocytes of AIDS patients. AIDS Res. Hum. Ret-
roviruses 17, 443–452.
12. Trial, J., Rubio, J. A., Birdsall, H. H., Rodriguez-Barradas, M., Rossen,
R. D. (2004) Monocyte activation by circulating fibronectin fragments in
HIV-1-infected patients. J. Immunol. 173, 2190–2198.
13. Nockher, W. A., Bergmann, L., Scherberich, J. E. (1994) Increased solu-
ble CD14 serum levels and altered CD14 expression of peripheral blood
monocytes in HIV-infected patients. Clin. Exp. Immunol. 98, 369–374.
14. Thieblemont, N., Weiss, L., Sadeghi, H. M., Estcourt, C., Haeffner-Cavail-
lon, N. (1995) CD14lowCD16high: a cytokine-producing monocyte subset
which expands during human immunodeficiency virus infection. Eur. J.
Immunol. 25, 3418–3424.
15. Dunne, J., Feighery, C., Whelan, A. (1996) ?-2-Microglobulin, neopterin
and monocyte Fc ? receptors in opportunistic infections of HIV-positive
patients. Br. J. Biomed. Sci. 53, 263–269.
16. Pulliam, L., Gascon, R., Stubblebine, M., McGuire, D., McGrath, M. S.
(1997) Unique monocyte subset in patients with AIDS dementia. Lancet
17. Amirayan-Chevillard, N., Tissot-Dupont, H., Capo, C., Brunet, C., Dignat-
George, F., Obadia, Y., Gallais, H., Mege, J. L. (2000) Impact of highly
active anti-retroviral therapy (HAART) on cytokine production and
monocyte subsets in HIV-infected patients. Clin. Exp. Immunol. 120, 107–
18. Jaworowski, A., Ellery, P., Maslin, C. L., Naim, E., Heinlein, A. C., Ryan,
C. E., Paukovics, G., Hocking, J., Sonza, S., Crowe, S. M. (2006) Normal
CD16 expression and phagocytosis of Mycobacterium avium complex by
monocytes from a current cohort of HIV-1-infected patients. J. Infect. Dis.
19. Otani, I., Akari, H., Nam, K. H., Mori, K., Suzuki, E., Shibata, H., Doi, K.,
Terao, K., Yosikawa, Y. (1998) Phenotypic changes in peripheral blood
monocytes of cynomolgus monkeys acutely infected with simian immuno-
deficiency virus. AIDS Res. Hum. Retroviruses 14, 1181–1186.
20. Williams, K., Westmoreland, S., Greco, J., Ratai, E., Lentz, M., Kim, W. K.,
Fuller, R. A., Kim, J. P., Autissier, P., Sehgal, P. K., Schinazi, R. F.,
Bischofberger, N., Piatak, M., Lifson, J. D., Masliah, E., Gonzalez, R. G.
(2005) Magnetic resonance spectroscopy reveals that activated monocytes
contribute to neuronal injury in SIV neuroAIDS. J. Clin. Invest. 115,
21. Bissel, S. J., Wang, G., Trichel, A. M., Murphey-Corb, M., Wiley, C. A.
(2006) Longitudinal analysis of monocyte/macrophage infection in sim-
ian immunodeficiency virus-infected, CD8? T-cell-depleted macaques
that develop lentiviral encephalitis. Am. J. Pathol. 168, 1553–1569.
22. Hasegawa, A., Liu, H., Ling, B., Borda, J. T., Alvarez, X., Sugimoto, C.,
Vinet-Oliphant, H., Kim, W. K., Williams, K. C., Ribeiro, R. M., Lackner,
A. A., Veazey, R. S., Kuroda, M. J. (2009) The level of monocyte turnover
predicts disease progression in the macaque model of AIDS. Blood 114,
23. Ziegler-Heitbrock, H. W., Passlick, B., Flieger, D. (1988) The monoclonal
antimonocyte antibody My4 stains B lymphocytes and two distinct mono-
cyte subsets in human peripheral blood. Hybridoma 7, 521–527.
24. Grage-Griebenow, E., Lorenzen, D., Fetting, R., Flad, H. D., Ernst, M.
(1993) Phenotypical and functional characterization of Fc ? receptor I
(CD64)-negative monocytes, a minor human monocyte subpopulation
with high accessory and antiviral activity. Eur. J. Immunol. 23, 3126–3135.
25. Szeberenyi, J. B., Rothe, G., Pallinger, E., Orso, E., Falus, A., Schmitz, G.
(2000) Multi-color analysis of monocyte and dendritic cell precursor het-
erogeneity in whole blood. Immunobiology 202, 51–58.
26. Weber, C., Belge, K. U., von Hundelshausen, P., Draude, G., Steppich,
B., Mack, M., Frankenberger, M., Weber, K. S., Ziegler-Heitbrock, H. W.
(2000) Differential chemokine receptor expression and function in hu-
man monocyte subpopulations. J. Leukoc. Biol. 67, 699–704.
27. Grage-Griebenow, E., Zawatzky, R., Kahlert, H., Brade, L., Flad, H., Ernst,
M. (2001) Identification of a novel dendritic cell-like subset of
CD64(?)/CD16(?) blood monocytes. Eur. J. Immunol. 31, 48–56.
28. Geissmann, F., Jung, S., Littman, D. R. (2003) Blood monocytes consist
of two principal subsets with distinct migratory properties. Immunity 19,
29. Sunderkotter, C., Nikolic, T., Dillon, M. J., Van Rooijen, N., Stehling, M.,
Drevets, D. A., Leenen, P. J. (2004) Subpopulations of mouse blood
monocytes differ in maturation stage and inflammatory response. J. Im-
munol. 172, 4410–4417.
30. Auffray, C., Fogg, D., Garfa, M., Elain, G., Join-Lambert, O., Kayal, S.,
Sarnacki, S., Cumano, A., Lauvau, G., Geissmann, F. (2007) Monitoring
of blood vessels and tissues by a population of monocytes with patrolling
behavior. Science 317, 666–670.
31. Steppich, B., Dayyani, F., Gruber, R., Lorenz, R., Mack, M., Ziegler-Heit-
brock, H. W. (2000) Selective mobilization of CD14(?)CD16(?) mono-
cytes by exercise. Am. J. Physiol. Cell Physiol. 279, C578–C586.
32. Kapinsky, M., Torzewski, M., Buchler, C., Duong, C. Q., Rothe, G.,
Schmitz, G. (2001) Enzymatically degraded LDL preferentially binds to
CD14(high) CD16(?) monocytes and induces foam cell formation medi-
ated only in part by the class B scavenger-receptor CD36. Arterioscler.
Thromb. Vasc. Biol. 21, 1004–1010.
33. Kim, W. K., Alvarez, X., Fisher, J., Bronfin, B., Westmoreland, S.,
McLaurin, J., Williams, K. (2006) CD163 identifies perivascular macro-
phages in normal and viral encephalitic brains and potential precursors
to perivascular macrophages in blood. Am. J. Pathol. 168, 822–834.
34. Moniuszko, M., Kowal, K., Rusak, M., Pietruczuk, M., Dabrowska, M.,
Bodzenta-Lukaszyk, A. (2006) Monocyte CD163 and CD36 expression in
human whole blood and isolated mononuclear cell samples: influence of
different anticoagulants. Clin. Vaccine Immunol. 13, 704–707.
35. Wei, Q., Stallworth, J. W., Vance, P. J., Hoxie, J. A., Fultz, P. N. (2006)
Simian immunodeficiency virus (SIV)/immunoglobulin G immune com-
plexes in SIV-infected macaques block detection of CD16 but not cyto-
lytic activity of natural killer cells. Clin. Vaccine Immunol. 13, 768–778.
36. Kuwata, T., Kodama, M., Sato, A., Suzuki, H., Miyazaki, Y., Miura, T.,
Hayami, M. (2007) Contribution of monocytes to viral replication in ma-
Journal of Leukocyte Biology
Volume , MONTH 2010
caques during acute infection with simian immunodeficiency virus. AIDS
Res. Hum. Retroviruses 23, 372–380.
37. Lentz, M. R., Kim, W. K., Lee, V., Bazner, S., Halpern, E. F., Venna, N.,
Williams, K., Rosenberg, E. S., Gonzalez, R. G. (2009) Changes in MRS
neuronal markers and T cell phenotypes observed during early HIV in-
fection. Neurology 72, 1465–1472.
38. Zhu, T., Muthui, D., Holte, S., Nickle, D., Feng, F., Brodie, S., Hwangbo,
Y., Mullins, J. I., Corey, L. (2002) Evidence for human immunodeficiency
virus type 1 replication in vivo in CD14(?) monocytes and its potential
role as a source of virus in patients on highly active antiretroviral ther-
apy. J. Virol. 76, 707–716.
39. Ellery, P. J., Tippett, E., Chiu, Y. L., Paukovics, G., Cameron, P. U., So-
lomon, A., Lewin, S. R., Gorry, P. R., Jaworowski, A., Greene, W. C.,
Sonza, S., Crowe, S. M. (2007) The CD16? monocyte subset is more per-
missive to infection and preferentially harbors HIV-1 in vivo. J. Immunol.
40. Valledor, A. F., Borras, F. E., Cullell-Young, M., Celada, A. (1998) Tran-
scription factors that regulate monocyte/macrophage differentiation.
J. Leukoc. Biol. 63, 405–417.
41. Nagamura-Inoue, T., Tamura, T., Ozato, K. (2001) Transcription factors
that regulate growth and differentiation of myeloid cells. Int. Rev. Immu-
nol. 20, 83–105.
42. Gemelli, C., Montanari, M., Tenedini, E., Zanocco, M. T., Vignudelli, T.,
Siena, M., Zini, R., Salati, S., Tagliafico, E., Manfredini, R., Grande, A.,
Ferrari, S. (2006) Virally mediated MafB transduction induces the mono-
cyte commitment of human CD34? hematopoietic stem/progenitor cells.
Cell Death Differ. 13, 1686–1696.
43. Kansas, G. S., Muirhead, M. J., Dailey, M. O. (1990) Expression of the
CD11/CD18, leukocyte adhesion molecule 1, and CD44 adhesion mole-
cules during normal myeloid and erythroid differentiation in humans.
Blood 76, 2483–2492.
44. Wood, B. (2004) Multicolor immunophenotyping: human immune sys-
tem hematopoiesis. Methods Cell Biol. 75, 559–576.
45. Whitelaw, D. M. (1972) Observations on human monocyte kinetics after
pulse labeling. Cell Tissue Kinet. 5, 311–317.
46. Al Izzi, S. A., Maxie, M. G., Valli, V. E., Wilkie, B. N., Johnson, J. A.
(1982) The kinetics of mononuclear phagocytes in normal calves given
Corynebacterium parvum. Can. J. Comp. Med. 46, 138–145.
47. Arenson Jr., E. B., Epstein, M. B., Seeger, R. C. (1980) Volumetric and
functional heterogeneity of human monocytes. J. Clin. Invest. 65, 613–
48. Yasaka, T., Mantich, N. M., Boxer, L. A., Baehner, R. L. (1981) Functions
of human monocyte and lymphocyte subsets obtained by countercurrent
centrifugal elutriation: differing functional capacities of human monocyte
subsets. J. Immunol. 127, 1515–1518.
49. Figdor, C. G., Bont, W. S., Touw, I., de Roos, J., Roosnek, E. E., De Vries,
J. E. (1982) Isolation of functionally different human monocytes by coun-
terflow centrifugation elutriation. Blood 60, 46–53.
50. Akiyama, Y., Miller, P. J., Thurman, G. B., Neubauer, R. H., Oliver, C.,
Favilla, T., Beman, J. A., Oldham, R. K., Stevenson, H. C. (1983) Charac-
terization of a human blood monocyte subset with low peroxidase activ-
ity. J. Clin. Invest. 72, 1093–1105.
51. Lewis, C. E., McCarthy, S. P., Lorenzen, J., McGee, J. O. (1989) Hetero-
geneity among human mononuclear phagocytes in their secretion of ly-
sozyme, interleukin 1 and type-? transforming growth factor: a quantita-
tive analysis at the single-cell level. Eur. J. Immunol. 19, 2037–2043.
52. Inamura, N., Sone, S., Okubo, A., Singh, S. M., Ogura, T. (1990) Hetero-
geneity in responses of human blood monocytes to granulocyte-macroph-
age colony-stimulating factor. J. Leukoc. Biol. 47, 528–534.
53. Wang, S. Y., Mak, K. L., Chen, L. Y., Chou, M. P., Ho, C. K. (1992) Het-
erogeneity of human blood monocyte: two subpopulations with different
sizes, phenotypes and functions. Immunology 77, 298–303.
54. Passlick, B., Flieger, D., Ziegler-Heitbrock, H. W. (1989) Identification
and characterization of a novel monocyte subpopulation in human pe-
ripheral blood. Blood 74, 2527–2534.
55. Bruno, L., Seidl, T., Lanzavecchia, A. (2001) Mouse pre-immunocytes as
non-proliferating multipotent precursors of macrophages, interferon-pro-
ducing cells, CD8?(?) and CD8?(–) dendritic cells. Eur. J. Immunol. 31,
56. Mizuno, K., Toma, T., Tsukiji, H., Okamoto, H., Yamazaki, H., Ohta, K.,
Ohta, K., Kasahara, Y., Koizumi, S., Yachie, A. (2005) Selective expansion
of CD16highCCR2– subpopulation of circulating monocytes with prefer-
ential production of haem oxygenase (HO)-1 in response to acute in-
flammation. Clin. Exp. Immunol. 142, 461–470.
57. Moniuszko, M., Bodzenta-Lukaszyk, A., Kowal, K., Lenczewska, D., Dab-
rowska, M. (2009) Enhanced frequencies of CD14??CD16?, but not
CD14?CD16?, peripheral blood monocytes in severe asthmatic patients.
Clin. Immunol. 130, 338–346.
58. Pauza, C. D., Galindo, J., Richman, D. D. (1988) Human immunodefi-
ciency virus infection of monoblastoid cells: cellular differentiation deter-
mines the pattern of virus replication. J. Virol. 62, 3558–3564.
59. Patterson, B. K., Mosiman, V. L., Cantarero, L., Furtado, M., Bhatta-
charya, M., Goolsby, C. (1998) Detection of HIV-RNA-positive monocytes
in peripheral blood of HIV-positive patients by simultaneous flow cyto-
metric analysis of intracellular HIV RNA and cellular immunophenotype.
Cytometry 31, 265–274.
60. Roulston, A., D’Addario, M., Boulerice, F., Caplan, S., Wainberg, M. A.,
Hiscott, J. (1992) Induction of monocytic differentiation and NF-? B-like
activities by human immunodeficiency virus 1 infection of myelomono-
blastic cells. J. Exp. Med. 175, 751–763.
61. Abel, P. M., McSharry, C., Galloway, E., Ross, C., Severn, A., Toner, G.,
Gruer, L., Wilkinson, P. C. (1992) Heterogeneity of peripheral blood
monocyte populations in human immunodeficiency virus-1 seropositive
patients. FEMS Microbiol. Immunol. 5, 317–323.
62. van der Kuyl, A. C. van den Burg, R., Zorgdrager, F., Groot, F., Berkhout,
B., Cornelissen, M. (2007) Sialoadhesin (CD169) expression in CD14?
cells is upregulated early after HIV-1 infection and increases during dis-
ease progression. PLoS ONE 2, e257.
63. Durrbaum-Landmann, I., Kaltenhauser, E., Flad, H. D., Ernst, M. (1994)
HIV-1 envelope protein gp120 affects phenotype and function of mono-
cytes in vitro. J. Leukoc. Biol. 55, 545–551.
64. Zembala, M., Bach, S., Szczepanek, A., Mancino, G., Colizzi, V. (1997)
Phenotypic changes of monocytes induced by HIV-1 gp120 molecule and
its fragments. Immunobiology 197, 110–121.
65. Freedman, B. D., Liu, Q. H., Del Corno, M., Collman, R. G. (2003)
HIV-1 gp120 chemokine receptor-mediated signaling in human macro-
phages. Immunol. Res. 27, 261–276.
66. Gessani, S., Borghi, P., Fantuzzi, L., Varano, B., Conti, L., Puddu, P., Be-
lardelli, F. (1997) Induction of cytokines by HIV-1 and its gp120 protein
in human peripheral blood monocyte/macrophages and modulation of
cytokine response during differentiation. J. Leukoc. Biol. 62, 49–53.
67. Koutsonikolis, A., Haraguchi, S., Brigino, E. N., Owens, U. E., Good,
R. A., Day, N. K. (1997) HIV-1 recombinant gp41 induces IL-10 expres-
sion and production in peripheral blood monocytes but not in T-lympho-
cytes. Immunol. Lett. 55, 109–113.
68. Dudhane, A., Wang, Z. Q., Orlikowsky, T., Gupta, A., Wormser, G. P.,
Horowitz, H., Kufer, P., Hoffmann, M. K. (1996) AIDS patient monocytes
target CD4 T cells for cellular conjugate formation and deletion through
the membrane expression of HIV-1 envelope molecules. AIDS Res. Hum.
Retroviruses 12, 893–899.
69. McElrath, M. J., Steinman, R. M., Cohn, Z. A. (1991) Latent HIV-1 infec-
tion in enriched populations of blood monocytes and T cells from sero-
positive patients. J. Clin. Invest. 87, 27–30.
70. Gibellini, D., Borderi, M., De Crignis, E., Cicola, R., Cimatti, L., Vitone,
F., Chiodo, F., Re, M. C. (2008) HIV-1 DNA load analysis in peripheral
blood lymphocytes and monocytes from naive and HAART-treated indi-
viduals. J. Infect. 56, 219–225.
subsets ? macrophage ? CD16 ? HIV ? AIDS
Kim et al.
Monocyte heterogeneity in SIV infection
Volume , MONTH 2010
Journal of Leukocyte Biology 11