The many faces of macrophage activation

David M. Mosser
Cell Biology and Molecular Genetics, University of Maryland, College Park
Key Words: immunoregulation autoimmunity IFN- IL-4
alternatively activated macrophage type 2-activated macrophage

THE CLASSICALLY ACTIVATED

MACROPHAGE

INTRODUCTION

As Yogi Berra might say, lets start from the beginning. It is

important to remember that macrophages become classically
activated by exposure to two signals. The first is the obligatory
cytokine IFN-, which primes macrophages for activation but
does not in itself activate macrophages [7]. The second signal
is tumor necrosis factor (TNF) itself or an inducer of TNF.
Exogenous TNF can act as the second signal, but the physiologically relevant second signal is generally the result of Tolllike receptor (TLR) ligation, which induces endogenous TNF
production by the macrophage itself. Thus, classically activated macrophages are developed in response to IFN-, along
with exposure to a microbe or microbial product such as LPS.
In the murine system, these cells are now easily identified by
virtue of their production of nitric oxide (NO) [8, 9]. Macrophages that have been primed with IFN- alone should not
make NO, provided the system is free of LPS (a common
contaminant in recombinant IFN-). In the human system, the
activation of macrophages is somewhat more difficult to definitively determine, as monocyte-derived macrophages from peripheral blood generally do not produce NO in response to the
classical activating stimuli. These cells must be identified by a
variety of biochemical and functional criteria (Table 1), including the up-regulation of surface molecules such as MHC
class II and B-7 (CD86) and an enhanced ability to present
antigen and kill intracellular pathogens. With the application
of microarrays, the analysis of classically activated macrophages has entered into a new era of complexity. Remarkably,
25% of the observed genes exhibited alterations in their expression upon macrophage activation [10]. The problem now
becomes selecting which of the several hundred genes that are
induced or suppressed during activation are true and reliable
markers of macrophage activation.
The biological functions of the activated macrophage are
myriad and well documented. These cells migrate to sites of
inflammation where they encounter pathogens and degrade
them. Contrary to popular belief, activated macrophages are
not more phagocytic than resting cells [11]. Although activated
macrophages spread out more than resident cells and generally
have a higher pinocytic rate, they express reduced levels of
mannose receptor and Fc receptor for IgG (FcR)II [12].
Activated macrophages do however possess a markedly enhanced ability to kill and degrade intracellular microorgan-

It used to be easy. In the old days (8 years ago), activated

macrophages were simply defined as cells that secreted inflammatory mediators and killed intracellular pathogens. Things are
becoming progressively more complicated in the world of leukocyte biology. Activated macrophages may be a more heterogenous
group of cells than originally appreciated, with different physiologies and performing distinct immunological functions. The first
hint of this heterogeneity came with the characterization of the
alternatively activated macrophage [1]. The exposure of macrophages to interleukin (IL)-4 or glucocorticoids induced a population of cells that up-regulated certain phagocytic receptors but
failed to produce nitrogen radicals [2] and as a result, were
relatively poor at killing intracellular pathogens. Recent studies
have shown that these alternatively activated cells produce several
components involved in the synthesis of the extracellular matrix
(ECM) [3], suggesting their primary role may be involved in tissue
repair rather than microbial killing. It turns out that the name
alternatively activated macrophage may be unfortunate for a few
reasons. First, although these cells express some markers of
activation, they have not been exposed to the classical, activating
stimuli, interferon- (IFN-) and lipopolysaccharide (LPS). Second, and more importantly, the name implies that this is the only
other way to activate a macrophage. Recent studies suggest that
this may not be the case. Exposure of macrophages to classical
activating signals in the presence of immunoglobulin G (IgG)
immune complexes induced the production of a cell type that was
fundamentally different from the classically activated macrophage. These cells generated large amounts of IL-10 and as a
result, were potent inhibitors of acute inflammatory responses to
bacterial endotoxin [4]. These activated macrophages have been
called type 2-activated macrophages [5] because of their ability to
induce T helper cell type 2 (Th2) responses that were predominated by IL-4 [6], leading to IgG class-switching by B cells. Thus,
at this time, there appears to be at least three different populations
of activated macrophages with three distinct biological functions.
The first and most well described is the classically activated
macrophage whose role is as an effector cell in Th1 cellular
immune responses. The second type of cell, the alternatively
activated macrophage, appears to be involved in immunosuppression and tissue repair. The most recent addition to this list is the
type 2-activated macrophage, which is anti-inflammatory and
preferentially induces Th2-type humoral-immune responses to
antigen. Together, these three populations of cells may form their
own regulatory network to prevent a well-intentioned immune
response from progressing to immunopathology.

Correspondence: David M. Mosser, Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742. E-mail: dm268@umail.umd.edu
Received June 25, 2002; accepted October 1, 2002; doi: 10.1189/jlb.0602325

Journal of Leukocyte Biology Volume 73, February 2003 209

TABLE 1.

Comparison of Distinct Murine Macrophage Subpopulations

Classical

Alternative

Type II

Activating signals

IFN-, TNF

IL-4 or glucocorticoids

TLR lignost, IgG complexes

TLR ligation

Secretory products

1TNF, 1IL-12
IL-1, IL-6

1IL-1RA
IL-10

1IL-10
TNF, IL-6

Biological markers

1MHC class II
1CD86
2Mannose receptor

1Mannose receptor
1Scavenger receptor
1CD23
2CD14
CD163
MS-1

1MHC class II
1CD86
(unique markers not yet available)

Killer molecules

NO, O2,

None

*NO, O2

Chemokine production

IP-10
MIP-1
MCP-1

AMAC-1

Unknown

* Although these cells can make NO and O2 in direct response to activating stimuli, the production of high levels of IL-10 by type II-activated macrophages
inhibits neighboring cells from responding to IFN- activation and making reactive nitrogen intermediates. MHC, Major histocompatibility complex; MS-1, MS-1
high-molecular-weight protein; IP-10, IFN-inducible protein 10; AMAC-1, alternative macrophage activation-associated CC-chemokine-1; MIP-1, macrophageinflammatory protein-1; MCP-1, monocyte chemoattractant protein-1.

isms, and for several years, this was the functional criterion
used to define an activated macrophage. This killing is accomplished by an increase in the production of toxic oxygen
species and an induction of the inducible NO synthase (iNOS)
gene to produce NO. The restriction of various nutrients from
the phagosome is an underappreciated but important aspect of
microbial killing that is also used by activated macrophages.
The restriction of iron [13] and tryptophan [14] from vacuolar
organisms is a particularly well-established mechanism for
limiting intracellular growth within activated macrophages.
Tissue macrophages can be seen in organized collections of
cells called granulomas, where they are frequently surrounded by
T cells producing activating cytokines. These granuloma macrophages are a rich source of inflammatory cytokines, toxic radicals,
and inflammatory lipid mediators. These products are capable of
causing extensive tissue damage to the host. For example, the
observation that tumors frequently developed proximal to old
tuberculosis scars [15] illustrates the destructive and mutagenic
potential of the activated macrophages that are frequently found
there. The immunopathology associated with many type 1 autoimmune diseases has the activated macrophage as the main
protagonist [16]. Thus, the activity of these cells must be carefully
regulated to prevent uncontrolled tissue destruction. Signals to
down-regulate macrophage activation may come from within the
macrophage itself or from surrounding cells. The immunoregulatory cytokines transforming growth factor (TGF)- and IL-10 play
an important role in dampening macrophage activation. Knockout
mice lacking either of these two cytokines have proven to be
hypersusceptible to inflammatory pathologies [17, 18]. A recently
described family of macrophage receptor tyrosine kinases in the
stem cell-derived tyrosine kinase receptor (STK/RON) family has
been implicated in the inhibition of macrophage activation [19].
The ligation of RON with macrophage stimulatory protein appears
to induce arginase activity (see below) and inhibit NO production.
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Journal of Leukocyte Biology Volume 73, February 2003

Thus, the ligation of this family of receptors may alter the phenotype of these cells into ones that resemble the alternatively activated macrophages described below. Finally, cell death by apoptosis cannot be ignored as an important immunoregulatory mechanism. The direct removal of inflammatory cells as well as the
induction of anti-inflammatory TGF- following apoptotic cell
removal [20] contribute to this effect.
One of the most interesting, new areas of inquiry in this field
is determining how intracellular pathogens interfere with signaling to prevent macrophage activation. The protozoan parasite Leishmania spp [21] and Mycobacterium spp [22] have
provided the best examples of such interference. Infection of
macrophages with either of these organisms prevents the macrophage from becoming fully activated in response to IFN-.
The molecules and mechanisms by which these organisms
circumvent cellular immunity will likely reveal important information about the process of macrophage activation itself.

THE ALTERNATIVELY ACTIVATED

MACROPHAGE
In the early 1990s, while examining the regulation of mannose
receptor expression on elicited macrophages, Gordon and colleagues [1] noticed that a particularly efficient way to induce
receptor expression was to treat these macrophages with IL-4.
The authors concluded that macrophages treated with IL-4
assumed an alternative activation phenotype. This represents the first description of a macrophage that exhibited signs
of activation but was clearly distinct from its classically activated counterpart. Subsequent studies by Goerdt and Orfanos
[23] have championed this cell as a regulatory macrophage
with diverse biological roles different from the classically
activated cell. These cells fail to make NO by virtue of their
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induction of arginase [24] and consequently, are compromised

in their ability to kill intracellular microbes. Although they
up-regulate some MHC class II molecules, they are not efficient at antigen presentation, and in many instances, they
actually inhibit T cell proliferation. The suppressive activity of
these macrophages extends to mitogen-activated T cells, which
show a significantly diminished proliferative and secretory
response in the presence of alternatively activated macrophages [25]. The signature cytokines produced by these alternatively activated macrophages are IL-10 and IL-1 receptor
antagonist. In the lung, it is thought that alternatively activated
macrophages may provide negative regulatory signals to protect
the host from overzealous inflammatory responses to environmental stimuli [26]. Recent studies on this cell population have
begun to focus on their potential to mediate wound-healing,
angiogenesis, and ECM deposition. Alternatively activated
macrophages produce high levels of fibronectin and a matrixassociated protein, IG-H3 [3], and they promote fibrogenesis
from fibroblastoid cells [27]. The induction of arginase in these
cells may lead to polyamine and proline biosynthesis, promoting cell growth, collagen formation, and tissue repair [28].
The in vivo schistosome infection model has proven to be an
invaluable one to unravel the function of altered macrophage
metabolism caused by type 2 cytokines [28]. Unlike many of the
autoimmune models mentioned above, in the Schistosome model,
type 2 immune responses correlate with pathology, whereas type 1
responses can ameliorate tissue destruction. This model has revealed that a competition for arginine metabolism within macrophages may be a key determinant of macrophage physiology.
When macrophages are treated with type 1 cytokines, the iNOS2
enzyme produces NO from arginine. When macrophages are exposed to type 2 cytokines, Arg-1 is induced and metabolizes
arginine to urea and ornithine, a precursor of polyamines and
proline. Polyamines are involved in cell growth and division,
whereas proline is a key component of collagen. In this elegant
model system, Wynn and colleagues [28] demonstrated unequivocally that Arg-1 levels correlate directly with Schistosome egginduced pathology, whereas iNOS levels inversely correlate with
pathology. Recently, macrophages from the (Th2-prone) BALB/c
mice were shown to make more arginase than macrophages from
the Th1-prone C57Bl/6 mice [29]. Thus, the alternatively activated macrophage may serve more of a regulatory and recovery
function than the effector killing functions that are associated with
classically activated macrophages.
Although the alternatively activated macrophage has been
investigated for only slightly more than 5 years, there exists a
solid battery of markers available to identify this cell type
(Table 1). In addition to arginase induction, these cells make a
signature chemokine, originally named AMAC-1 [30]. They
react well with monoclonal antibodies to a specific form of
CD163 and an uncharacterized MS-1 antigen. Finally, two
transcription factors, FIZZ1 and Ym1, appear to be preferentially induced in IL-4-treated macrophages [31].

THE TYPE II-ACTIVATED MACROPHAGE

The type II-activated macrophage was initially identified during an examination of conditions under which IL-12 transcrip-

tion was terminated. It was observed that the ligation of FcRs

on activated macrophages unexpectedly turned off IL-12 synthesis [32] and induced the secretion of large amounts of IL-10
[33]. A variety of immune complexes and a number of different
activating stimuli were used, and in all cases, FcR ligation
resulted in a specific and reciprocal alteration in the production of these two cytokines [4]. Similar to the classically
activated macrophages described above, the type II activation
phenotype required two signals. The first signal was the ligation of FcRs. However, receptor ligation had to be coupled
with a macrophage stimulatory signal to influence cytokine
production. Stimuli included those that signal through any of
the TLRs, as well as through CD40 or CD44. The type II
phenotypic switch following FcR ligation worked on
unprimed and IFN--primed macrophages. Type II-activated
macrophages are clearly distinct from alternatively activated
macrophages described above, as they do not induce arginase,
and with the exception of IL-12 and IL-10, the production of
many of the other cytokines produced by classically activated
macrophages, such as TNF, IL-1, and IL-6, remains intact [4].
The dramatic induction of IL-10 by these macrophages
suggested that they would have anti-inflammatory properties.
To test this, an acute model of LPS lethality was implemented.
Type II-activated macrophages were generated in vitro and
transferred into mice, which then received six times the lethal
dose (90) of LPS. Mice that received 1 106 type II macrophages remained completely viable and healthy throughout the
observation period, whereas mice that received control macrophages succumbed to lethal endotoxemia within 48 h [4]. To
show that this effect was a result of IL-10 secretion, type
II-activated macrophages from IL-10-/- mice were used in
parallel. These macrophages failed to rescue mice from lethal
endotoxemia. Thus, by virtue of their secretion of IL-10, the
type II-activated macrophage exerts a potent anti-inflammatory
effect that can be exploited to prevent acute pathologies, such
as those associated with LPS endotoxemia.
The name, type II-activated macrophages, was derived from
their ability to preferentially induce Th2 adaptive immune
responses. In the initial in vitro studies, classically or type
II-activated macrophages were used as antigen presenting cells
(APC). Classically activated macrophages stimulated T cells to
produce primarily IFN- in response to antigen, whereas type
II-activated macrophages induced T cells to produce high
levels of IL-4 [6]. This bias in T cell cytokine production by
these two populations of APC was stable and preserved when T
cells were subsequently restimulated under a variety of nonbiasing conditions. T cell cytokine production correlated with
APC cytokine production. IL-12 secretion by classically activated macrophages induced IFN- production by T cells,
whereas IL-10 secretion by type II-activated macrophages
resulted in IL-4 production by T cells [6].
To determine the extent to which type II-activated macrophages could influence antibody responses to antigen, mice
were injected with type II- or classically activated macrophages along with ovalbumin (OVA) antigen in the absence of
any other adjuvant. Mice vaccinated with OVA in the presence
of classically activated macrophages made only modest IgGantibody responses to antigen [5]. Mice vaccinated with OVA
in the presence of type II-activated macrophages, however,
Mosser Macrophage activation

211

made significantly more IgG, and the majority of the IgG was of
the IgG1 isotype. This observation suggested that the presence
of type II-activated macrophages alone was sufficient to induce
(Th2-like) IgG class-switching. Thus, the ligation of FcR on
activated macrophages by antigen-IgG complexes induced T
cells to produce IL-4, which in turn induced B cells to produce
IgG1 in response to that antigen.
These studies demonstrate that antigen binding to the FcR on
activated macrophages will preferentially induce a Th2-like response. Thus, they predict that IgG itself can be a strong inducer
of humoral immunity and an inhibitor of cell-mediated immunity
by virtue of its ability to change the phenotype of the APC. These
observations may have important, practical implications, as they
suggest that reagents that ligate the FcR on activated macrophages may have anti-inflammatory properties that can be exploited to ameliorate autoimmune diseases. These studies may
also have particular relevance to autoimmunity, where activated
macrophages likely represent important APCs.

11.
12.

13.
14.
15.
16.
17.
18.
19.

SUMMARY
Macrophage heterogeneity used to be a research topic on which
the careers of many postdoctoral fellows were misspent. The lack
of definitive markers and dubious biochemical assays prevented
the unequivocal identification of specific cell subsets. There has
now been significant progress in establishing the heterogeneity of
activated macrophages. There are at least three distinct populations of macrophages, and each cell type appears to have different
biological roles. The interplay among these populations of cells
may help to shape not only the magnitude but also the character
of the immune response. The manipulation of these cells may lead
to new approaches to treat or prevent disease.

20.
21.

22.
23.
24.
25.

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