© 2005 Nature Publishing Group
Circulating monocytes give rise to a variety of tissue-
resident macrophages throughout the body, as well as
to specialized cells such as dendritic cells (DCs) and
OSTEOCLASTS. Monocytes are known to originate in the
bone marrow from a common myeloid progenitor that
is shared with neutrophils, and they are then released
into the peripheral blood, where they circulate for
several days before entering tissues and replenishing
the tissue macrophage populations1. The morphology
of mature monocytes in the peripheral circulation is
heterogeneous, and these cells constitute ∼5–10% of
peripheral-blood leukocytes in humans. They vary
in size and have different degrees of granularity and
varied nuclear morphology, which at the extremes
of variation can lead to confusion with granulocytes,
lymphocytes, natural killer cells and DCs. The basic
features of the mononuclear-phagocyte system (which
includes macrophages and their monocyte precursors
and lineage-committed bone-marrow precursors, as
well as all other cells that are derived from this lineage2)
are summarized in BOX 1.
As long ago as 1939, Ebert and Florey3 reported
the observation that monocytes emigrated from blood
vessels and developed into macrophages in the tissues.
Pro-inflammatory, metabolic and immune stimuli all
elicit increased recruitment of monocytes to peripheral
sites4, where differentiation into macrophages and DCs
occurs, contributing to host defence, and tissue remod-
elling and repair. Studies of the mononuclear-phagocyte
system, using monoclonal antibodies specific for vari-
ous cell-surface receptors and differentiation antigens,
have shown that there is substantial heterogeneity of
phenotype, which most probably reflects the special-
ization of individual macrophage populations within
their microenvironments. Although it is clear that
monocytes are precursors of both macrophage and DC
lineages, this development and differentiation pathway
is still relatively poorly studied in vivo. However, the
identification of heterogeneity among peripheral-blood
monocytes — first, in humans, and more recently, in
mice — has provided a powerful insight into the nature
of myeloid-cell heterogeneity and has provided novel
ways to assess cell fate and function in vivo.
The cellular and molecular adhesion mechanisms
that are used by migrating monocytes have recently
been reviewed5 and are not discussed here. Instead,
we briefly summarize the current understanding of
human monocyte heterogeneity and then describe
recent advances in the study of mouse monocyte
heterogeneity, to highlight the different physiological
Sir William Dunn School
of Pathology, University of
Oxford, South Parks
Road, Oxford OX1 3RE, UK.
Correspondence to S.G.
A multinucleate cell that
MONOCYTE AND MACROPHAGE
Siamon Gordon and Philip R. Taylor
Abstract | Heterogeneity of the macrophage lineage has long been recognized and, in part,
is a result of the specialization of tissue macrophages in particular microenvironments.
Circulating monocytes give rise to mature macrophages and are also heterogeneous
themselves, although the physiological relevance of this is not completely understood.
However, as we discuss here, recent studies have shown that monocyte heterogeneity is
conserved in humans and mice, allowing dissection of its functional relevance: the different
monocyte subsets seem to reflect developmental stages with distinct physiological roles,
such as recruitment to inflammatory lesions or entry to normal tissues. These advances in
our understanding have implications for the development of therapeutic strategies that are
targeted to modify particular subpopulations of monocytes.
NATURE REVIEWS | IMMUNOLOGY
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© 2005 Nature Publishing Group
Adult bone marrow
Adult tissues Adult peripheral blood
AFFERENT LYMPHATIC VESSEL
A vessel that carries lymph into
a lymph node.
roles of the subsets in vivo, and we discuss the relevance
of heterogeneity to the study of human monocyte biol-
ogy. We also discuss the origins of tissue-resident mac-
rophage populations, and we describe how knowledge
of their origins might be pertinent to tissue homeostasis
at times when it could be advantageous to modulate
monocyte function as part of a therapeutic strategy.
Monocyte heterogeneity in humans
Peripheral-blood monocytes show morphological
heterogeneity, such as variability of size, granularity
and nuclear morphology. Monocytes were initially
identified by their expression of large amounts of
CD14 (which is part of the receptor for lipopoly-
saccharide). However, the subsequent identifica-
tion of differential expression of antigenic markers
showed that monocytes in human peripheral blood
are hetero geneous, and this provided the first clues
to the differential physiological activities of monocyte
subsets. Differential expression of CD14 and CD16
(also known as FcγRIII) allowed monocytes to be
divided into two subsets: CD14hiCD16– cells, which
are often called classic monocytes, because this pheno-
type resembles the original description of monocytes;
and CD14+CD16+ cells6. It was subsequently shown
that the CD14+CD16+ monocytes expressed higher
amounts of MHC class II molecules and CD32
(also known as FcγRII), and it was suggested that
these cells resemble mature tissue macrophages7.
Distinct chemokine-receptor expression profiles
were also among the phenotypic differences that
were recognized between these subsets: for example,
CD14+CD16+ monocytes expressed CC-chemokine
receptor 5 (CCR5), whereas CD14hiCD16– monocytes
expressed CCR2 REF. 8. A summary of the cell-surface-
marker phenotype of these human monocyte subsets is
presented in TABLE 1.
When cultured, both human monocyte subsets can
differentiate into DCs in the presence of granulocyte/
macrophage colony-stimulating factor (GM-CSF) and
interleukin-4 (IL-4)9,10. Furthermore, using an in vitro
transendothelial-migration model, in which freshly
isolated peripheral-blood mononuclear cells are
incubated with human umbilical-vein endothelial-cell
monolayers grown on an endotoxin-free collagenous
matrix, it has been shown that monocytes can migrate
across an endothelial barrier in vitro and differentiate
either into macrophages, which remain in the sub-
endothelial matrix, or into DCs, which then migrate
back across the endothelial layer11. In this model, the
CD14+CD16+ monocyte subset was found to be more
likely to become DCs and reverse transmigrate than
was the CD14hiCD16– monocyte subset12, indicating
that the CD14+CD16+ cells might be precursors of
DCs, which can pass through tissues and then migrate
to the lymph nodes through the AFFERENT LYMPHATIC
VESSELS. However, these observations do not preclude an
in vivo role for CD14hiCD16– monocytes in contribut-
ing to the DC pool. Importantly, the extent to which
monocytes differentiate into macrophages or DCs
in this model depends on both the phenotype of the
cultured monocyte population and the factors that are
present in the culture. For example, transendothelial
migration of CD14+CD16+ monocytes can be induced
with soluble CX3C-chemokine ligand 1 (CX3CL1; also
Box 1 | The mononuclear-phagocyte system
Phagocytic cells were initially classified as the reticuloendothelial system87; however,
this classification failed to distinguish between ‘true’ sinusoidal endothelial cells and
sinus-lining macrophages. As a consequence, the classification of the mononuclear-
phagocyte system was altered to include only macrophages and their monocyte
precursors and lineage-committed bone-marrow precursors2. However, this
classification might require further refinement as the origins of fetal macrophages
become clearer and as unanswered questions about the common precursors of
macrophages and lymphocytes are addressed.
During development, the origins of cells from the yolk sac that have macrophage-
like phenotypes might be distinct from the origins of these cells in adults and after
haematopoiesis properly begins in the fetal liver88,89 (see figure). Developing
macrophages are first found in the yolk sac, as identified by morphological
characteristics, as well as by expression of macrophage markers such as FMS,
CD11b and the mannose receptor90–92. Studies in zebrafish, in which cell lineage
can more easily be followed because of the transparency of the embryos, have shown
population of the yolk sac with macrophage precursors; these cells then differentiate
and emigrate into the head mesenchyme and its circulation93. Later in development,
haematopoiesis in the fetal liver becomes a source of macrophages that resemble
those that are present in adults. Initially, haematopoiesis in the liver generates large
numbers of macrophages, which are present in most organs. Population of the organs
of the embryo with phagocytes is discussed elsewhere88,89. Furthermore, many recent
studies (for further details, see the main text) indicate that, although monocytes can
be precursors for the replenishment of tissue-resident macrophage populations,
many of these macrophages might be derived from local proliferation in the adult
rather than from recruited peripheral monocytes. When bone-marrow monocytes
are released into the peripheral blood (having a Ly6C+ phenotype), they are thought
to differentiate into a phenotypically distinct (Ly6C–) cell subset (for further details,
see the main text).
G-CFU, granulocyte colony-forming unit; GM-CFU, granulocyte/macrophage colony-forming
unit; HSC, haematopoietic stem cell; M-CFU, macrophage colony-forming unit.
954 | DECEMBER 2005 | VOLUME 5
© 2005 Nature Publishing Group
known as fractalkine) or CXC-chemokine ligand 12
(CXCL12; also known as SDF1α), the receptors for
which are preferentially expressed by these cells13.
An additional monocyte subset that is defined by the
expression of CD14, CD16 and CD64 (also known as
FcγRI) has been reported more recently14. These cells
seem to combine characteristics of monocytes and
DCs, with high expression of CD86 and HLA-DR
and high T-cell-stimulatory activity. Compared with
CD14hiCD16– (classic) monocytes (which are also
CD64+), these CD14+CD16+CD64+ cells have a simi-
larly high phagocytic activity and produce similarly
Table 1 | Phenotype of the two best-characterized monocyte subsets in various mammals*
CCR1+– NDND NDND ND ND
CCR4+– ND NDND NDND ND
CCR5–+ NDND NDNDND ND
CCR7+– ND ND+– NDND
CXCR1+– ND NDND NDNDND
CXCR2+– NDND NDND ND ND
CXCR4+++ NDND NDND ND ND
+ +++ ++–+ND ND
CD4++ NDND +/–++ NDND
CD11a ND ND+ ++NDND+++
CD11b++ ++++++ ++++ND ND
CD14 ++++ ND ND NDND +++
CD31+++ +++ +++ ND NDND ND
CD32 ++++ NDND ++++ ND ND
CD33 ++++ ND NDND ND ND ND
CD43ND ND–+–+ NDND
CD49bNDND+– NDND NDND
CD62L‡ ++–+–+– ND ND
CD80 NDND NDND NDND+ ++
CD86+ ++ND NDNDND+ ++
CD115 ++ ++ ++++ND ND ND ND
NDND ND ND
CD200RND NDND ND+– NDND
F4/80ND ND++ ND NDND ND
Ly6C NDND+– NDND NDND
7/4 ND ND+– ND NDND ND
MHC class II+ ++–– NDND+ ++
*‘Inflammatory’ and ‘resident’ nomenclature is based on studies carried out in mice and extrapolated to other species. Data have been assigned arbitrary symbols that
represent no expression (–), marginal expression (+/–) and increasing amounts of expression (+, ++, +++). Mouse monocyte-subset phenotypic data are derived from
REFS 16,22,25,35,94,95. Rat monocyte data were kindly provided by U. Yrlid (personal communication). Other data are taken from the references cited in the main text.
For a more detailed definition of the cell-surface receptor and functional heterogeneity of human monocytes, see REF. 15. ‡These receptors show good conservation
of expression-pattern differences between the subsets of at least three species. §There is no available commercial reagent that recognizes the extracellular domain of
mouse CD116 (also known as granulocyte/macrophage colony-stimulating-factor receptor α-chain). 7/4, an unidentified mouse antigen recognized by monoclonal
antibody 7/4; CCR, CC-chemokine receptor; CD200R, CD200 receptor; CXCR, CXC-chemokine receptor; CX3CR1, CX3C-chemokine receptor 1; EMR1, epidermal-
growth-factor-module-containing mucin-like hormone receptor 1; F4/80, monoclonal antibody that recognizes the mouse homologue of the human protein EMR1;
ND, either differences between the subsets have not been determined or, in some cases, there is no clear species conservation of the antigens.
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MIXED LEUKOCYTE REACTION
A tissue-culture technique for
testing T-cell reactivity. The
proliferation of one population
of T cells, induced by exposure
to inactivated MHC-
mismatched stimulator cells, is
determined by measuring the
incorporation of 3H-thymidine
into the DNA of dividing cells.
A liposome that contains the
liposomes are ingested by
macrophages, resulting in cell
A liposome that is labelled
with the fluorochrome Dil
perchlorate). These liposomes
are internalized by phagocytic
cells, rendering the cells
large amounts of cytokines (such as tumour-necrosis
factor (TNF) and IL-6), and these phenotypes are
not shared with the CD14+CD16+CD64– subset.
However, the CD14+CD16+CD64+ cells share with
the CD14+CD16+CD64– subset a greater stimulatory
activity in MIXED LEUKOCYTE REACTIONS than CD14hiCD16–
monocytes. A summary of the phenotypic properties
of human monocyte subsets, such as the reduced
phagocytic activity and increased stimulatory capac-
ity in mixed leukocyte reactions has been compiled
by Grage-Griebenow et al.15 The authors speculated
that, although the origins of this CD14+CD16+CD64+
subset are not known, it could be an immunoregulatory
monocyte phenotype or, possibly (because of the simi-
lar characteristics of these cells to both monocytes and
DCs), an intermediate phenotype between monocytes
So, although these observations from the past 20
years have provided insights into the fate and func-
tion of human monocyte subsets, the restriction
of the study of monocyte heterogeneity to in vitro
analyses of human cells and the initial failure to
recog nize the mouse counterparts of these monocyte
subsets hampered determination of the functional
roles of the monocyte subsets in a physiological
Monocyte heterogeneity in mice
Antigenic differentiation of two monocyte subsets
in mice was first achieved after the observation that
monocytes (identified in mice by their F4/80+CD11b+
phenotype) could be subdivided according to their
expression of CCR2, CD62L (also known as L-selectin)
and CX3C-chemokine receptor 1 (CX3CR1; measured
by expression of green fluorescent protein (GFP) in
cells from mice in which GFP had been ‘knocked-in’
to one of the Cx3cr1 alleles)16. One monocyte subset
expressed CCR2, CD62L and only moderate amounts
of CX3CR1, whereas the second did not express CCR2
or CD62L but expressed higher amounts of CX3CR1.
The CCR2+ monocyte subset was, as expected, found
to migrate towards the CCR2 ligand CC-chemokine
ligand 2 (CCL2; also known as MCP1)16. The expres-
sion of CCR2 and the capacity to migrate towards
CCL2 is consistent with the important role of this
chemokine and its receptor in the recruitment of
monocytes to inflammatory lesions, so the subset
of monocytes that expresses CCR2 in mice is known as
the ‘inflammatory’ subset17–21. Dan Littman’s research
group extended our knowledge of the phenotypic
differences between the two monocyte subsets that
have been described for mice and humans, and they
paid particular attention to the expression pattern of
chemokine receptors TABLE 1. As can be seen from
TABLE 1, the receptors that are differentially expressed
by the inflammatory-monocyte subset are broadly
considered to be chemokine and adhesion receptors
that are involved in the recruitment of leukocytes
to an inflammatory lesion. In addition, Geissmann
et al.22 identified Ly6C (which is part of the epitope
of GR1) as an additional marker of CCR2+ monocytes
in mice TABLE 1. These studies indicated that CCR2+
CD62L+CX3CR1lowLy6C+ mouse monocytes corre-
spond to CD14hiCD16– (classic) human monocytes,
which are also CCR2+CX3CR1low and that CCR2–
CD62L–CX3CR1hiLy6C – mouse monocytes correspond
to CD14+CD16+CD64 – human monocytes, which also
express large amounts of CX3CR1. These observations
were the first to indicate that it would be possible to
address the in vivo relevance of human monocyte
heterogeneity by studying mice.
Functional characterization of mouse monocyte
subsets. To distinguish functionally between the two
mouse monocyte subsets, Geissmann and colleagues22
adoptively transferred GFP-expressing monocytes
from the Cx3cr1-knock-in mice and studied cell fate
and function in naive and immunologically challenged
recipient mice. They found that the CCR2+CX3CR1low
monocytes were short-lived after adoptive transfer and
were difficult to detect in the tissues of naive recipi-
ents. However, as anticipated by their expression of
CCR2 and CD62L (molecules that are known to be
involved in inflammatory-cell recruitment17–21,23,24),
these cells were rapidly recruited to sites of experi-
mentally induced inflammation22 (the reason for the
term inflammatory monocyte). After migration into
the inflamed site, the CCR2+ monocytes upregulated
expression of CD11c and MHC class II molecules,
and some CD11c+MHC class II+ cells were recovered
from the draining lymph nodes, indicating that they
might have differentiated into DCs. Experimental
evidence for the ability of CCR2+ monocytes to dif-
ferentiate into DCs was also obtained by transferring
CCR2+ monocytes into MHC-class-I-deficient mice
and showing that the transferred cells could prime
naive CD8+ T cells22.
By contrast, the CCR2–CX3CR1hi monocyte
population was found to persist for longer in animals
after adoptive transfer, and it was possible to recover
GFP-marked cells from the blood, spleen, lungs, liver
and brain of recipients for several days after transfer.
Some of the donor cells that were recovered from the
spleen had acquired a DC-like phenotype (that is,
CD11c+MHC class II+) following transfer. Similar to
their human counterparts9,10, both mouse monocyte
subsets have been reported to differentiate into DCs
in vitro when cultured with GM-CSF and IL-4 REF. 22.
However, the observation that the CCR2– monocytes
can enter tissues and acquire a DC-like phenotype
under steady-state conditions is consistent with the
finding that their human counterparts (CD14+CD16+
monocytes) preferentially differentiate into DCs in the
in vitro transendothelial-migration model12.
It was not clear at which point the division in lin-
eage between the two monocyte subsets occurred.
Sunderkotter and colleagues25 attempted to address this
question with a series of experiments based on in vivo
depletion of all monocytes with CLODRONATELOADED LIPO
SOMES and in vivo labelling with DILLABELLED LIPOSOSOMES,
followed by study of the repopulation kinetics of both
monocyte subsets. After depletion, the first monocytes
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Bone marrowPeripheral bloodInflamed tissue
Draining lymph node
Kupffer cells, alveolar
to reappear in the circulation were the CCR2+ (inflam-
matory) subset. The phenotype of these cells resembles
that of monocytes found in the bone marrow26, and
their appearance at this stage is consistent with the
hypothesis that these monocytes precede the CCR2–
subset in the developmental pathway. Unfortunately,
the adoptive transfer of purified CCR2+ monocytes did
not formally confirm this hypothesis, although this was
attributed to inappropriate manipulation of the mono-
cyte subset during purification, because the transferred
cells could not be recovered from the circulation25. In
addition, Sunderkotter and colleagues25 identified a
rare population of monocytes that were character-
ized by intermediate expression of Ly6C. Assuming
that the proposed pathway of CCR2–Ly6C– monocyte
derivation from CCR2+Ly6C+ monocytes is true, this
population might be an intermediate phenotypic state
between the two subsets (FIG. 1).
It had already been shown that inflammatory
monocytes recruited to the skin after injection of
fluorescent latex beads could ingest the beads, and
although most of these cells remained in the tissue as
macrophages, others could migrate to the T-cell area
of the draining lymph node and acquire a DC-like
phenotype (that is, CD11c+MHC class II+)27. While
investigating the possible role of CCR2 and CX3CR1
in regulating the migration of latex-bead-carrying
monocytes from the skin to the draining lymph
nodes, proliferation of Ly6Cmid monocytes, associ-
ated with a relative reduction in the size of the Ly6C+
monocyte subset, was observed in the peripheral
blood of CCR2-deficient mice28. Genetic deficiency
in CCR2 or CX3CR1 did not reduce the migration
of the latex-bead-carrying DC-like cells to the
lymph nodes; however, an unexpected increase in
migrating latex-bead-carrying cells was observed
in CCR2-deficient animals. This was reflected by
an increase in the proportion of latex-bead-carrying
Ly6Cmid cells in the skin of CCR2-deficient animals
after the injection of latex beads, indicating that the
recruitment of these cells to the skin did not depend
on CCR2 in this model. The authors speculated that the
increase in the proportion of these cells in the peripheral
blood and inflamed skin of CCR2-deficient mice and
in the number of monocyte-derived DC-like cells in
the draining lymph node might be linked and that the
Figure 1 | Development and function of monocyte subsets in mice. Ly6C+ bone-marrow monocytes are released into
the peripheral blood (a) and are thought to adopt a Ly6Cmid phenotype (b), which is associated with selective expression of
CC-chemokine receptor 7 (CCR7) and CCR8 and retention of CCR2, before (under steady-state conditions) they form
CCR2–Ly6C– monocytes (c) that are characterized by high CX3C-chemokine receptor 1 (CX3CR1) expression25,28. Both Ly6C+
and Ly6Cmid monocytes respond to pro-inflammatory cues, such as the CCR2 ligand CC-chemokine ligand 2 (CCL2), and are
recruited to inflammatory lesions22,28 (d). Most ‘inflammatory’ monocytes are thought to differentiate into macrophages, which are
important for clearance of pathogens and for the resolution of inflammation (e). Some monocytes emigrate from the tissues to the
draining lymph nodes, a process that uses CCR7 and CCR8 receptor–ligand interactions28 (f). CCR7+CCR8+ monocytes that are
present in the tissue must be recruited directly from the peripheral blood to the inflammatory site or must differentiate from Ly6C+
monocytes in situ (or must arise from a combination of both of these mechanisms). The expression of CCR7 and CCR8 by these
cells makes them uniquely disposed to emigrate into the lymphatic vessels. In the draining lymph nodes, these monocytes
acquire dendritic cell (DC)-like characteristics (g), which they do not obtain if they are retained in the tissue by selective
chemokine-receptor deficiency28. In the absence of inflammation, CX3CR1hiLy6C– monocytes enter the tissues and replenish the
tissue-resident macrophage and DC populations22 (h). Solid arrows represent pathways that are supported by established data,
whereas dashed arrows represent pathways that are indicated from a compilation of more recent data and speculation.
CX3CL1, CX3C-chemokine ligand 1.
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The membrane that lines
the abdominal cavity.
Inflammation of the
peritoneum that is induced by
sterile injection of an irritant,
such as thioglycollate broth.
This results in the sequential
recruitment of granulocytes,
monocytes and lymphocytes.
It is widely used to study acute
EFFERENT LYMPHATIC VESSEL
A vessel that carries lymph out
of a lymph node.
Ly6Cmid monocytes could be a subset of monocytes
that is predisposed to differentiate into DCs28. Ly6Cmid
monocytes have also been suggested to resemble
Ly6C– monocytes rather than Ly6C+ monocytes in
terms of their capacity to stimulate allogeneic cells28.
Reverse-transcription-PCR analysis of the three sub-
sets indicated that Ly6Cmid monocytes express higher
amounts of the mRNAs that encode CCR7 and CCR8
than do the other two monocyte subsets but similar
amounts of mRNA that encodes CCR2 to that of
Ly6C+ monocytes. Furthermore, analysis of mice that
are deficient in CCR8 or the ligands of CCR7 showed
a reduction in the number of latex-bead-carrying cells
with DC-like phenotype that migrated to the draining
lymph node, supporting a role for these molecules
in this process. Parallel in vitro studies with human
monocytes showed that blockade of CCR8 consider-
ably reduced the reverse transmigration of human
monocytes without affecting the initial migration
across the endothelial layer28.
The results of these studies of the heterogen eity
of mouse (and human) monocytes are summa-
rized in FIG. 1. Briefly, by extrapolation from the
current data, a picture is emerging in which bone-
marrow-derived monocytes (with the phenotype
CCR2+CX3CR1lowLy6C+) are released into the circula-
tion and, in the absence of inflammation, alter their
functional and phenotypic characteristics, passing
through an intermediate phenotype (CCR2+CCR7+
CCR8+Ly6Cmid). Both the bone-marrow-derived
monocytes and the monocytes of intermediate pheno-
type can respond to pro-inflammatory cues, migrate
to inflamed tissues and differentiate into macrophages
and DCs. The monocytes with an intermediate pheno-
type might be particularly predisposed to migrate to
the draining lymph nodes and differentiate into DCs.
In the absence of inflammation, a switch in monocyte
phenotype, the mechanism of regulation of which is
unknown, generates monocytes that are postulated
to enter the tissues and replenish the tissue-resident
macrophage and DC populations; these are known
as the ‘resident’ monocyte population (which has the
phenotype CCR2–CX3CR1hiLy6C–). The similarities
between human and mouse monocyte subsets indicate
that this is a conserved system, and further analysis of
the mouse system and other mammalian systems will be
helpful for understanding human monocyte biology.
Monocyte heterogeneity in other mammals
The study of monocyte heterogeneity in mice, in the
context of transgenic and gene-knockout technology, is a
powerful tool to investigate the mechanisms of monocyte
function and fate, but it also has limitations. Constraints
in working with mice, such as the limited availability
of cells and the limited accessibility of physiological
systems to physical manipulation and/or intervention,
merit the extension of these studies to larger mammalian
models. In this context, heterogeneity of monocytes has
been reported in rats and in pigs, with notable parallels
evident between these species and the observations that
have been made for mice and humans TABLE 1.
Rats. Monocyte heterogeneity was first shown in
rats by using CD43 as a differential marker29. In rats,
CD43hi monocytes express higher amounts of CD4
than do CD43low monocytes, and the CCR2–CX3CR1hi
monocyte subset in mice has also been shown to
express CD43 REF. 25. Recently, rat monocytes have
been further characterized antigenically and func-
tionally (U. Yrlid, personal communication). CD43low
monocytes express higher amounts of CCR2, CCR7,
CD32 and CD62L than do CD43hi monocytes, and they
migrate to the PERITONEUM during experimental STERILE
PERITONITIS, confirming their similarity to CCR2+Ly6C+
(inflammatory) mouse monocytes. By contrast, CD43hi
monocytes express higher amounts of CX3CR1 and
CD11c than do CD43low monocytes, so this popula-
tion is analogous to the Ly6C– (resident) monocyte
population in mice.
The use of rats has facilitated two important
experiments that were technically more demanding
in mice. First, when adoptively transferred, puri-
fied CCR2+CX3CR1low rat monocytes intravenously
acquired the phenotype of CCR2–CX3CR1hi mono-
cytes, strongly supporting the lineage development
that was previously proposed to occur in mice25.
Second, a model of mesenteric lymphadenectomy
and thoracic-duct cannulation, in which afferent and
EFFERENT LYMPHATIC VESSELS fuse, allowed the collection of
migrating DCs. Following adoptive transfer of CCR2–
CX3CR1hi monocytes, some donor cells acquired
the phenotype of intestinal-lymph DCs without the
requirement for additional stimulation, indicating that
the transferred resident-monocyte population could
differentiate into DCs under steady-state conditions.
Importantly, both the conversion of CCR2+CX3CR1low
monocytes into CCR2–CX3CR1hi monocytes and the
differentiation of CCR2–CX3CR1hi monocytes into cells
with the phenotype of intestinal-lymph DCs occurred
without cell division.
Pigs. Monocyte heterogeneity has also been described
in pigs30. CD163– monocytes express higher amounts of
CD14 than do their CD163+ counterparts, which in turn
express higher levels of MHC class II molecules, several
adhesion molecules (such as CD11a and CD11c) and
the co-stimulatory molecules CD80 and CD86; CD163+
monocytes also have a higher allostimulatory activity
and a greater ability to present soluble antigen to T cells
than do CD163– monocytes30,31. Both subsets of mono-
cyte can also differentiate into DCs when cultured in
the presence of GM-CSF and IL-4 REF. 32. The greater
allostimulatory activity and co-stimulatory molecule
expression by the CD163+ monocyte subset indicates
that these cells might correspond to Ly6C– (resident)
monocytes in mice.
Origins of macrophages and DCs
Tissue macrophages have a broad role in the mainte-
nance of tissue homeostasis, through the clearance of
senescent cells and the remodelling and repair of tissues
after inflammation33,34. They are generally considered
to be derived from circulating monocytes and show a
958 | DECEMBER 2005 | VOLUME 5
© 2005 Nature Publishing Group
A type of receptor that binds
conserved molecular structures
that are found in pathogens.
Examples include the mannose
receptor, which binds
terminally mannosylated and
and Toll-like receptors, which
are activated by various
microbial products, such as
hypomethylated DNA, flagellin
and double-stranded RNA.
A cell-surface receptor that is
involved in the internalization
of selected polyanionic ligands,
including modified low-density
A type of macrophage that is
present in the splenic white
pulp and is involved in the
clearance of apoptotic cells.
The connective tissue that
underlies the epithelium of the
gut mucosa. It contains various
myeloid and lymphoid cells,
dendritic cells, T cells and
A professional antigen-
presenting dendritic cell that is
localized in the epidermal layer
of the skin.
An individual that has received
a transplant of bone marrow
from another individual.
Mice that share a circulatory
system as a result of surgical
A committed precursor in
haematopoietic tissues that
can form granulocytes and
macrophages in the presence
of specific growth factors.
high degree of heterogeneity, which has largely been
uncovered through studies with monoclonal antibod-
ies35–37. The heterogeneity reflects the specialization of
function that is adopted by macrophages in different
anatomical locations, including the following: the abil-
ity of osteoclasts to remodel bone38; the high expression
of PATTERNRECOGNITION RECEPTORS and SCAVENGER RECEPTORS
by alveolar macrophages39–41, which are involved in
clearing microorganisms, viruses and environmental
particles in the lungs; and the positioning of thymic
macrophages42 and TINGIBLEBODY MACROPHAGES43 in the
germinal centre for clearance of apoptotic lympho-
cytes that are generated during the development of an
acquired immune response. The gut is one of the rich-
est sources of macrophages in the body, and isolation
of macrophages from the LAMINA PROPRIA has highlighted
a unique macrophage phenotype that is characterized
by high phagocytic and bactericidal activity but weak
production of pro-inflammatory cytokines. This pheno-
type can be induced in peripheral-blood-derived macro-
phages by intestinal stromal-cell products, indicating
that the tissue microenvironment can markedly influ-
ence the phenotype of tissue-resident macrophages44.
In addition to macrophage heterogeneity in different
organs, macrophage heterogeneity can be observed in a
single organ, and the mouse spleen, which is discussed
briefly in this section, is a particularly good example
Since the recent unravelling of monocyte hetero-
geneity, immediate attention has focused on studies
of monocyte fate, mainly regarding the question of
the potential of monocytes to form DCs in vitro and
in vivo and the possible effect of this on the immune
response. By contrast, relatively little attention has been
given to the perhaps more challenging question of the
role of monocyte heterogeneity in the generation of
macrophage populations in mice and the nature of the
circulating precursors of macrophage populations.
For example, it is still not clear whether tissue macro-
phages are derived from particular lineage-committed
precursors or whether they are derived randomly from
the monocyte pool. In addition (and particularly
in the case of inflammation-elicited macrophages),
after cells have ‘differentiated’ in a microenvironment,
are the cells then terminally differentiated, or are they
functionally flexible and able to alter their phenotype
in response to changes in their location? As previously
mentioned, most macrophages in the tissues of an adult
are considered to be derived from circulating mono-
cytes, which constitutively replenish tissue-resident
macrophage populations. However, studies of the ori-
gins of many tissue-resident macrophage populations
have shown that local proliferation has a considerable
role in the renewal and maintenance of many macro-
phage types (particularly under steady-state condi-
tions), with the recruitment of circulating precursors
having little, if any, role in this process in some cases.
But inflammatory insults, such as trauma or infection,
can lead to an increased dependence on the recruit-
ment of blood-borne precursors to aid repopulation
of the tissue-resident populations in many of these
inflamed tissues. Van Furth et al.45 summarized the
results of the initial studies in this area, although these
questions have been re-investigated more recently,
leading to the need to modify some of the older ideas
about the cellular origins of macrophages.
A good understanding of the origins of these cells,
as well as the timing and context of their recruitment,
will be essential not only to understand how tissues
recover from exposure to pro-inflammatory stimuli
but also to determine how these cells help to restore
the status quo ante, which is important for diseases
in which a loss of tissue homeostasis might result
from dysfunction of the tissue-resident macrophage
population(s). Examples in which the origins of
tissue-resident macrophage populations have been
investigated are discussed in this section and are
illustrated in FIG. 2. We have concentrated on the adult
because of the substantial differences in the develop-
ment of phagocytes in the adult and the embryo, which
are briefly summarized in BOX 1. One aspect that is
clear in many of these examples is that the nature of
the circulating precursor that is involved in the renewal
of tissue-resident populations remains poorly defined.
Studies such as those that were outlined in previous
sections for determining monocyte fate during inflam-
mation might be useful for studying the potential
involvement of individual monocyte subsets in the
renewal of tissue-resident cells.
Langerhans cells. LANGERHANS CELLS populate the epider-
mis during ontogeny and were originally thought to be
constantly replenished by bone-marrow-derived cells.
However, it has recently been shown that Langerhans
cells are self-renewing 46. Using BONEMARROW CHIMERAS
made with congenic cells (which were followed for
18 months) and PARABIOTIC MICE (which were followed
for 6 months), it was shown that Langerhans cells
were solely of recipient origin for the duration of these
studies46. When Langerhans cells were depleted by
irradiation with ultraviolet light, they were replenished
from circulating bone-marrow-derived precursors in
a CCR2-dependent manner, indicating that a circulat-
ing CCR2+ precursor is only utilized when the system
is under stress46. Similar observations have been made
in a case of human hand-allograft transplantation. At
4.5 years after transplantation, the Langerhans cells
were solely of donor origin, supporting the idea that
replacement of Langerhans cells by recipient bone-
marrow precursors is rare under steady-state condi-
tions47. Whether the CCR2-dependent recruitment of
the Langerhans-cell precursor involves recruitment
of an inflammatory monocyte or a lineage-committed
precursor is unclear.
Osteoclasts. Osteoclast precursors are found in the
GRANULOCYTE/MACROPHAGE COLONYFORMING UNIT GMCFU
PRECURSOR population and can be derived from unfrac-
tionated, mature monocytes from peripheral blood48–50.
Osteoclast precursors express the receptor for macro-
phage colony-stimulating factor (M-CSF; also known
as CSF1) and depend on it for their development.
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a Normal epidermis
d Alveolar space
e Central nervous system
b Inflamed epidermis
Stromal cells or osteoblasts
(Mcsfop/Mcsfop mice). An inbred
strain of mice that suffers from
osteopetrosis (stony bones) as
a result of deficient function
of osteoclasts. The defect has
been localized to the gene that
encodes macrophage colony-
stimulating factor (M-CSF; also
known as CSF1).
OSTEOPETROTIC MICE (Mcsfop/Mcsfop mice) have a defect
in osteoclast development51, and this has been shown
to result from a naturally occurring mutation in the
gene that encodes M-CSF52, underlining the impor-
tance of M-CSF in osteoclast development. Mice with
a disrupted Rankl (receptor-activator-of-nuclear-
factor-κB ligand) gene also have osteopetrosis53, and
this is consistent with the proposed role of RANKL
in osteoclast development54. Culture of unfraction-
ated peripheral-blood monocytes with M-CSF and
RANKL is sufficient to induce their differentiation
into osteoclasts, and it has been assumed that osteo-
clast precursors are monocytes, although this has not
been shown in vivo.
Alveolar macrophages. Alveolar macrophages have
been reported to be derived both from precursors
in peripheral blood and from local proliferation of
precursors. Early experiments in which radiosensitive
precursors of monocytes in mouse bone marrow were
transiently depleted with 89Sr — resulting in the loss
of peripheral-blood monocytes without affecting the
Figure 2 | Origins of mature cells in the periphery in adult mice. The importance of circulating precursors in the
repopulation of tissue-resident macrophage and dendritic cell (DC) populations is not fully understood. Furthermore, the
nature of the precursor — that is, whether it is a peripheral-blood monocyte or a circulating lineage-committed precursor —
is unclear. In the case of Langerhans cells, local proliferation in the normal epidermis (a) is adequate to maintain Langerhans-
cell numbers without the requirement for replenishment by circulating precursors; however, after inflammation (b), the
CC-chemokine receptor 2 (CCR2)-dependent recruitment of precursors to the epidermis allows repopulation of the tissue-
resident populations. In bone (c), it is thought that circulating precursors are recruited to the bone surface, where — under
the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator-of-nuclear-factor-κB ligand (RANKL)
— they differentiate into mature osteoclasts, which are multinucleate and resorb bone. In the lungs (d), alveolar
macrophages can be sustained for long periods by local proliferation; however, bone-marrow-transplantation experiments
show that blood-borne precursors can repopulate and divide in the lungs. In the central nervous system (e), microglia are
similarly maintained by local proliferation after an initial embryonic phase that populates the central nervous system, but
peripheral-blood monocytes also contribute to this pool. In the spleen (f), there is marked heterogeneity of macrophages,
with red-pulp, white-pulp, marginal-zone and metallophilic macrophages occupying specific anatomical niches. A simplified
schema is shown, which also indicates B cells, DCs and endothelial cells. At present, splenic macrophages are thought to
be replenished by a mixture of emigration from the circulating monocyte pool and local proliferation. GM-CSF, granulocyte/
macrophage colony-stimulating factor.
960 | DECEMBER 2005 | VOLUME 5
© 2005 Nature Publishing Group
A disease that is caused by
accumulation of surfactant
proteins in the alveoli.
A type of macrophage that is
derived from bone marrow,
arborized and present in the
parenchyma of the central
A type of macrophage that
lines small blood vessels: for
example, near the surface of the
A type of macrophages that is
present in the meninges (the
three membranes that surround
A type of macrophage that is
present at the interface between
the blood and the cerebrospinal
fluid in the brain.
A type of macrophage that is
present in the splenic marginal
zone and is involved in the
recognition and clearance of
material, such as pathogen-
derived material, from the
A type of macrophage that
surrounds the splenic white
pulp, adjacent to the marginal
number of alveolar macrophages — supported a role
for local proliferation; however, these experiments
allowed only short-term studies55. Further support
for local proliferation of precursors being the main
source of alveolar macrophages under normal condi-
tions came from radiation-chimera studies in mice: if
the lungs were protected from radiation, then most
alveolar macrophages were of recipient origin almost
1 year after treatment56. However, after whole-body
irradiation and transfer of GFP-labelled bone marrow,
host alveolar macrophages were replaced with cells
of donor origin over a long period, indicating that
alveolar macrophages can be replenished from the
bone marrow57. Studies in humans who have received
allogeneic bone-marrow transplants support this argu-
ment and indicate that this replenishment occurs by
recruitment of precursors, followed by proliferation of
these cells in situ58,59. The importance of GM-CSF in
lung physiology indicates that this growth factor has a
crucial role in the maintenance of alveolar-macrophage
populations, the maturation and/or activity of which
are impaired in GM-CSF-deficient mice, which suffer
from an ALVEOLARPROTEINOSIS-like disease60,61. Although it
is assumed that a monocyte subset might be the precur-
sor of alveolar macrophages, this has not been directly
Macrophages in the central nervous system. The
central nervous system (CNS) contains various macro-
phage subsets, including MICROGLIA, PERIVASCULAR
MACROPHAGES, MENINGEAL MACROPHAGES and CHOROIDPLEXUS
MACROPHAGES. Meningeal macrophages are thought to be
rapidly replaced by cells of bone-marrow origin, whereas
the replacement of perivascular and choroid-plexus
macrophages is slower, which indicates that replacement
of the individual populations might occur through dif-
ferent mechanisms or might rely on a shared mecha-
nism to differing extents62. For example, microglial cells
seem to persist much longer than do other macrophages
in the CNS, and their origins have been investigated by
a combination of in vivo labelling of dividing cells and
bone-marrow-transplantation experiments. Microglial
cells can proliferate in situ, and this might be one of the
main sources of microglia in adults; however, bone-
marrow-derived cells can enter the CNS across the
blood–brain barrier and populate the microglial-cell
compartment63,64. Monocytes are assumed to be capable
of entering the CNS and differentiating into microglia.
Splenic macrophages. Aspects of the heterogen eity
of splenic macrophages have been summarized
elsewhere65,66, but we briefly highlight, in FIG. 2, the
discrete anatomical localization of the different macro-
phage populations. Macrophages in the white pulp
include tingible-body macrophages. MARGINALZONE
MACROPHAGES are found adjacent to the marginal sinus
(through which the circulation passes), and they
express pattern-recognition receptors and scavenger
receptors, which aid in the clearance of blood-borne
pathogens67–69. METALLOPHILIC MACROPHAGES are found
adjacent to the white pulp and marginal sinus and
can sample the circulation70; although their function
is unknown, they might be important during viral
infections71 and other infections. Several studies have
attempted to address the question of the origins of
these different macrophage populations, both under
steady-state conditions and after experimental deple-
tion of the tissue-resident cells72–75, which is important
because (as mentioned previously) repopulation after
experimental insult might differ from repopulation
that occurs in the steady state. It seems that local
proliferation does occur under steady-state condi-
tions, at least with respect to some macrophage
subsets, such as white-pulp macrophages and metallo-
philic macrophages. In addition, it seems that circu-
lating precursors also contribute, but the nature of
these precursors is unknown. Unlike in humans, the
spleen of adult mice is considered to be a haemato-
poietic organ, and it differs structurally from the
human spleen. This might mean that the mouse is
not an ideal model for the study of cellular origins
for comparison with humans. Studies with the
M-CSF-deficient mice (Mcsfop/Mcsfop mice) highlight
the different dependencies of certain macrophage sub-
sets on M-CSF for their survival, because these mice
lack metallophilic macrophages while maintaining
reasonable numbers of most other splenic macrophage
Kupffer cells. Kupffer cells are an important compo-
nent of the mononuclear-phagocyte system that is
present in the liver. The origin of Kupffer cells has
been speculated to involve two mechanisms: replen-
ishment by local proliferation, and recruitment of
circulating precursors. Twelve hours after administration
of 3H-thymidine, ∼1.5% of Kupffer cells incorporate
3H-thymidine, giving an indication of the low number
of cells that are proliferating under steady-state condi-
tions in the adult79. The proportion of Kupffer cells that
incorporate 3H-thymidine increases if mice are exposed
to whole-body irradiation; however, shielding of the
hind legs of the animals during whole-body irradiation
indicates that the increase in 3H-thymidine incorpora-
tion depends on bone-marrow precursors79. In mouse
bone-marrow transplants, donor-derived cells rapidly
populate the liver with Kupffer cells (within 3 weeks),
and donor Kupffer cells in liver transplants are replaced
with similar kinetics80. However, experiments in rats
indicate that Kupffer cells might be long-lived81, and
temporary depletion of peripheral-blood monocytes
with 89Sr had little effect on Kupffer-cell numbers80. So,
Kupffer cells, similar to many other macrophage popu-
lations, can be replenished by distinct mechanisms, and
it is probable that the mechanisms used are affected by
inflammation and other factors.
Inflammatory-monocyte-derived macrophages. It
has long been recognized that inflammatory mono-
cytes (now defined as CCR2+Ly6C+ monocytes) are
recruited and differentiate into macrophages at the site
of the inflammatory lesion4. A series of in vitro experi-
ments has led to macrophages often being ascribed
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VOLUME 5 | DECEMBER 2005 | 961
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Peripheral blood Inflamed tissue
Innate activation by TLR ligands
(for example, LPS, LTA and PGN)
Increased production of pro-inflammatory
cytokines, iNOS and ROS
Classical activation by IFN-γ γ and LPS
Increased production of pro-inflammatory
cytokines, iNOS and ROS; increased
expression of MHC class II molecules and
CD86; increased antigen presentation; and
increased microbicidal activity
Alternative activation by IL-4 or IL-13
Increased endocytic activity; increased
expression of mannose receptor, dectin-1
and arginase; increased cell growth; increased
tissue repair; and increased parasite killing
Deactivation by IL-10, TGF-β β,
CD200–CD200R, CD47– CD172a or steroids
Increased production of IL-10, TGF-β and PGE2;
and reduced expression of MHC class II molecules
activation states66,82–84 (FIG. 3). These include the
following: classical activation, which can be induced
by in vitro culture of macrophages with interferon-γ
and lipopolysaccharide (which induces TNF pro-
duction) and is associated with high microbicidal
activity, pro-inflammatory cytokine production
and cellular immunity; alternative activation, which
results from culture in IL-4 or IL-13 and is associ-
ated with tissue repair and humoral immunity; innate
activation, which is mediated in culture by ligation of
receptors such as Toll-like receptors (most of which
are expressed by cells of the monocyte–macrophage
lineage85) and is associated with microbicidal activity
and pro-inflammatory cytokine production; deacti-
vation, which is induced by culture in the presence
of cytokines such as IL-10 or transforming growth
factor-β, or by ligation of inhibitory receptors such
as CD200 receptor or CD172a, and is associated with
anti-inflammatory cytokine production and reduced
MHC class II expression. Despite these classifications,
the extent of plasticity in this system is unclear: that
is, it is unclear whether macrophage fate is deter-
mined once or whether it is constantly malleable, and
it is also unclear whether distinct ‘activation states’
exist in vivo or whether macrophages, instead, show
a broad range of phenotypes. It is probable that, in
most situations, an inflammatory environment
leads to the exposure of macrophages to multiple
stimuli, with complex phenotypic consequences.
The recent advances in our ability to follow the fate
of the monocyte lineage in vivo (such as the identi-
fication and adoptive transfer of monocyte subsets),
in conjunction with single-cell analysis, could be
applied to these fundamental questions of macro-
phage behaviour and fate during inflammation. It
would be particularly useful to study situations such
as granuloma formation and parasitic infections, in
which polarization of macrophage activation has been
implicated in disease pathology and in which a strong
bias in the nature of the immune response has been
observed in vivo.
The study of animal models has led to a rapid advance
in our understanding of the functional consequences
of monocyte heterogeneity, and this knowledge can be
directly applied to the study of human biology. The
ever-expanding pool of genetically manipulated mod-
els will accelerate the progression of knowledge in this
field. The initial similarities between the species that
have been studied reinforce that there is a conserved
commonality in the function of these systems, thereby
validating their use. Further study will be important to
understand how monocytes are recruited to particular
inflammatory sites and what determines their differen-
tiation into DCs or macrophages, cell populations that
regulate acquired immune responses and/or carry out
surveillance of normal and abnormal tissues.
Until now, the characterization of monocyte
heterogeneity has largely been led by hypothesis-driven
investigations and has been restricted to candidate
approaches, such as the extensive use of chemokine-
receptor-deficient mice in experimental models of
inflammation. Advances in the isolation of monocyte
subsets, as well as the increasing availability of improved
DNA-microarray and proteomic analysis, should
provide important and more objective insights into the
extent of the cellular diversification of function that is
present in the monocyte lineage.
Another important issue will be how this current
research translates into the advancement of medi-
cal knowledge. Techniques for imaging macrophage
recruitment and accumulation in humans (for example,
in patients with atherosclerosis) have involved transfer
of labelled autologous monocytes or in vivo uptake of
fluorodeoxyglucose by metabolically active cells,
which (although not specific) correlates with macro-
phage numbers and plaque stability 86. More selective
strat egies have been adopted in animal models, such
as the in vivo administration of radiolabelled chemo-
kines, which would be anticipated to bind selectively to
certain subsets of monocytes (among other cells)86.
Perhaps a more challenging aspect of these studies is
to determine the contribution of monocytes, or alterna-
tive, lineage-committed precursors, to the macrophage
and DC populations of the body. It is clear that, for the
development of appropriate therapeutic interventions,
a better understanding of the natural cell lineage of
monocytes is required, because long-term modulation
of monocyte activities could have implications for the
maintenance of tissue populations of macrophages and
DCs and therefore could affect homeostasis, immunity
Figure 3 | Macrophage heterogeneity during inflammation. In inflamed tissues, the
precursors of the elicited macrophages — that is, ‘inflammatory’ peripheral-blood monocytes
— are now better understood than was previously the case but are still not completely
understood. However, the ability of these elicited macrophages to acquire distinct phenotypes
and physiological activities has been observed in vitro. For example, when stimulated with
interferon-γ (IFN-γ), macrophages show high microbicidal activity and produce reactive oxygen
species (ROS). By contrast, when cultured with interleukin-4 (IL-4), IL-10, IL-13 or transforming
growth factor-β (TGF-β), a phenotype is generated that promotes tissue repair and suppresses
inflammation. Whether these phenotypes are distinct or whether they indicate a continuum of
physiological responsiveness remains unclear. CD200R, CD200 receptor; iNOS, inducible nitric-
oxide synthase; LPS, lipopolysaccharide; LTA, lipoteichoic acid; PGE2, prostaglandin E2;
PGN, peptidoglycan; TLR, Toll-like receptor; TNF, tumour-necrosis factor.
962 | DECEMBER 2005 | VOLUME 5
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Competing interests statement
The authors declare no competing financial interests.
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