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Review
The concept of an alternative pathway of macrophage activation has stimulated interest in its definition,
mechanism, and functional significance in homeostasis and disease. We assess recent research in this field,
argue for a restricted definition, and explore pathways by which the T helper 2 (Th2) cell cytokines interleukin-
4 (IL-4) and IL-13 mediate their effects on macrophage cell biology, their biosynthesis, and responses to
a normal and pathological microenvironment. The stage is now set to gain deeper insights into the role of
alternatively activated macrophages in immunobiology.
Introduction tion factor STAT6 in their signaling (Heller et al., 2008). These
The concept of an alternative pathway of macrophage activation and other experiments showed that these Th2 cytokines were
induced by the T helper 2 (Th2) cytokines interleukin-4 (IL-4) and not simply inhibitory, but subtle modulators of macrophage func-
IL-13, distinct from interferon-g (IFN-g)-mediated classical acti- tions. We proposed that IL-4 induced an ‘‘alternative’’ form of
vation, has gained considerable ground over the past decade. activation in macrophages (Stein et al., 1992).
Analysis of marker molecules in the mouse has revealed a stereo- Subsequently, other workers introduced the term M1 and M2
typic signature of expressed genes induced by IL-4 and IL-13, to parallel macrophage activation with T helper cell polarization,
contrasting with characteristic changes induced by IFN-g, which subdivided M2 classes into subgroups, and in some cases,
mediates Th1 cell-type activation of macrophages. Phenotypes termed all nonclassical activation phenotypes as ‘‘alternative’’
associated with alternative activation of macrophages (AAMs) (discussed in Gratchev et al., 2001; Mantovani et al., 2008;
and classical activation of macrophages (CAMs) can be distin- Mosser and Edwards, 2008). We have argued that too loose
guished from the direct effects of microbial stimuli, such as lipo- a definition obscures the study of mechanism and discovery of
polysaccharide, which induce innate macrophage activation, potential therapeutic agents. Even a restricted definition of IL-4
often through Toll-like receptors (TLRs). Innate stimuli are able and IL-13 dependence needs to take into account the complex
to synergize with CAMs, to achieve full expression of macro- effects of cell differentiation, interactions with other receptors,
phage effector pathways. and signaling pathways, and the role of tissue microenvironment
Investigators have implicated AAMs in a range of physiologic in modulating the macrophage phenotype. Monocyte and mac-
and pathological processes, including homeostasis, inflamma- rophage heterogeneity and nomenclature are discussed below
tion, repair, metabolic functions, and malignancy. This is in addi- and elsewhere (Gabrilovich and Nagaraj, 2009; Geissmann
tion to their established role in immunity, hypersensitivity, allergy, et al., 2010).
parasitic infections, and their sequelae, such as fibrosis. Recent
genome-wide analysis, although broadly supporting earlier stud- Analytic Approaches
ies with limited numbers of markers, has underscored the There are two distinct, complementary approaches to the
heterogeneity and plasticity of macrophage gene expression, study of AAMs. The first, ‘‘forward analysis,’’ flows from recep-
adding to the confusing nomenclature of monocyte and tissue tor-dependent recognition, through signal transduction, gene
macrophage subpopulations (Geissmann et al., 2010). In this expression, and protein production. The second, ‘‘reverse anal-
review, we assess current knowledge, point to the need to ysis,’’ deduces from phenotypic markers the mechanism of initi-
restrict the concept of AAM, and consider newer findings ation. Studies with isolated macrophages and in situ analysis
regarding mechanisms and therapeutic potential. in wild-type and genetically modified mice made it possible to
Our own interest in IL-4 modulation of the macrophage pheno- combine these approaches. Forward analysis utilizes cytokine,
type arose in the late 1980s from experiments with human blood receptor, or signaling-deficient animals to define an AAM pheno-
monocyte-derived macrophages and mouse peritoneal macro- type, including Arginase 1 (ARG1), Ym1 and Ym2, Fizz1, MRC1,
phages. At the time, the role of this cytokine was believed to and selected chemokines (Loke et al., 2002). Signature markers,
be mainly anti-inflammatory, resulting from its ability to suppress in combination, have then been used to infer an AAM pathway
TNF and IL-6 production in macrophages. Whereas macrophage and mechanism (reverse analysis).
mannose receptor (MRC1) expression and its endocytic function There are obvious caveats to the use of reverse analysis alone,
were selectively downregulated by IFN-g (Mokoena and Gordon, as required in human studies in situ: substantial species differ-
1985), it was upregulated by IL-4 (Stein et al., 1992) and IL-13 ences exist in available markers, and established markers such
(Doyle et al., 1994) and depended in vitro and in vivo on as Arg1 can be induced by other pathways (El Kasmi et al.,
IL4Ra1, the common IL-4 and IL-13 receptor alpha chain (Line- 2008). Even clusters of multiple signature markers can be inde-
han et al., 2003). Others showed a critical role for the transcrip- pendent of the IL-4 and IL-13 receptor pathway (Daley et al.,
Review
2010; Stout, 2010). We lack markers for human AAM, but matory cytokines, such as IL-10 and TGF-b. Proinflammatory
these should become available with current microarray and cytokines include IL-1b, TNF, and IL-6. Through potent mem-
proteomic studies. Combined forward and reverse analysis brane protein assemblies they generate reactive oxygen, nitro-
in the mouse will validate the link between recognition and gen, and arachidonate metabolites.
response. With humans, our dilemma mimics the problem of The study of chromatin structure and epigenetic control of
diagnosis in clinical medicine, where a cluster of symptoms and gene expression in macrophages is in its infancy. Given the
signs cannot be assigned to a particular cause without sup- range of receptors and potential ligands, it may be invidious to
portive evidence. single out IFN-g, IL-4, and IL-13 receptor pathways as central
regulators of macrophage activation. However, given their often
Toward an Integrated Approach polarized production in the immune response, these contrasting
Before turning to the pathways and functions of AAM, we states of activation provide an important conceptual framework.
summarize general features of monocyte and macrophage Other receptors participate in forming signaling platforms and
heterogeneity (Gordon, 2008). Macrophages arise from hemo- complexes, e.g., G protein-coupled receptors and integrins.
poietic progenitors (Geissmann et al., 2010), which differentiate, Crosstalk at all levels impinges on altered transcription. It is
directly or via circulating monocytes, into subpopulations of therefore possible to conceive an infinite number of potential
tissue macrophages and closely related myeloid dendritic cells phenotypes; however, the conserved, stereotypic macrophage-
(Steinman and Idoyaga, 2010), the latter specialized to present specific signature for IFN-g versus IL-4- and IL-13-dependent
antigens to naive T helper lymphocytes. Osteoclasts arise from activation provides useful insights into physiologic and patho-
monocyte precursors to form multinucleated giant cells, impli- logic mechanisms.
cated in resorption of bone. Monocyte and macrophages share Figure 1 presents a schematic model of macrophage activa-
properties with granulocytes, especially neutrophils, and to tion. The first stage, differentiation, depends on growth factors
lesser extent with B lymphocytes. Monocyte and macrophages such as GM-CSF or M-CSF; the second, priming by IFN-g
migrate through several body compartments, including bone (CAMs) or IL-4 and IL-13 (AAMs); and the third, a localizing stim-
marrow, blood, lymphoid, and all nonhematopoietic tissues. ulus delivered by a TLR or analogous receptor. This additional
Resident macrophages are present in organs constitutively, in stimulus promotes induction of a full complement of classical
the absence of overt inflammation, and perform trophic as or alternative activation functions. In the case of AAMs, this
well as homeostatic roles in the removal of apoptotic cells, may arise in a Th2 cell-biased environment, e.g., chronic para-
serving as sentinels of injury and infection. Tissue macrophages sitic infection, followed by a secondary microbial challenge.
can replicate locally, but are terminally differentiated, turning Counteracting negative controls contribute to a fourth stage of
over at different rates, depending on the stimulus and tissue resolution and repair. Once established, some properties of full
environment. alternative activation can be reversible (plasticity) whereas
Macrophages differ morphologically and phenotypically in others may be irreversible (Stout et al., 2005).
organs such as liver, spleen, lung, gut, and brain, interacting Cytokines in AAM Induction
with matrix and other cell types. They are active in biosynthesis IL-4 and IL-13 are the prototypical direct inducers of AAMs, but
and express a wide range of receptors, recognizing foreign as other cytokines such as IL-33 and IL-25 amplify AAM induction
well as normal and abnormal cells and host-derived products. indirectly, through Th2 cells (for review on CD4+ T cell heteroge-
Through constitutive and induced endocytosis, phagocytosis, neity, see O’Shea and Paul, 2010). IL-4 and IL-13 are produced
and secretion of various products, including cytokines, growth by various innate cells, including non-B non-T cells (Moro et al.,
factors, and metabolites, they perform both trophic and toxic 2009; Neill et al., 2010), mast cells, basophils, eosinophils, NKT
functions, serving as a widely dispersed mononuclear phago- cells, and even macrophages themselves. Apart from activated
cyte system during development and throughout adult life. Th2 cells and B cells, epithelia and tumor cells are potential sour-
They contribute to tissue remodeling, host defense in innate ces in tissues. The availability of reporter mice has contributed to
and adaptive immunity, and many disease processes. the analysis of IL-4 production in vivo, during allergic inflamma-
Monocytes are the main source of newly recruited tissue tion and infection (Mohrs et al., 2005).
macrophages in infection, granulomata, atherosclerosis, Alz- The role of IL-5, another Th2 cell cytokine that binds to distinct
heimer’s disease, and tumors. Differentiation and activation of receptors on macrophages, is unclear. IL-10, produced by CD4+
macrophages depend on specific growth and differentiation Th2 cells, macrophages, and B cells as well as tumor cells, is
factors, their receptors, signaling pathways, and transcription a potent downregulator of macrophage gene expression, modu-
factors. Macrophages migrate to local sites of injury and infec- lating IL-4 and IL-13 and IFN-g actions. IL-21 enhances AAMs by
tion, contributing to acute and chronic inflammation, locally driving IL-4R expression (Pesce et al., 2006). IL-33, a Th2 cell
and systemically. In addition they acquire enhanced cytotoxic, product, binds to ST2L, a subunit of the IL-33 receptor, to
antimicrobial, and inhibitory activities, initiate repair, and resolve amplify Th2 cytokine production and AAM induction (Kurow-
inflammation. ska-Stolarska et al., 2009).
Macrophages express opsonic and a range of nonopsonic IL-25, a member of the IL-17 family also produced by Th2
receptors, TLRs, RIG-I-like and Nod-like sensing receptors, on cells, induces production of multipotent hemopoietic progeni-
their surface and in vacuolar and cytosolic compartments. Other tors in bone marrow and at sites of extramedullary hemopoiesis,
receptors include families of regulatory molecules, lectins, and giving rise to a range of myeloid cells, including monocytes and
scavenger receptors. Their secretory repertoire includes anti- macrophages (Saenz et al., 2010). IL-25 activates antigen-
bacterial and proteolytic enzymes, chemokines, and anti-inflam- specific responses in mucosal tissues, such as the colon. In
Review
Review
Figure 2. IL-4 and IL-13 Signaling
Type I Type II Machinery
IL-4 binds IL-4Ra and then recruits either IL-2Rg
IL-4 IL-13
chain (to form the type I receptor) or the IL-13Ra1
IL-4
chain (to make the type II receptor), depending
IL-13 upon the cell type (Heller et al., 2008). However,
IL-13 can only signal through type II receptor as
IL-4Rα IL-2Rγ IL-4Rα IL-13Rα1 IL-13Rα2 it binds IL-13Ra1 and recruits IL-4Ra (LaPorte
et al., 2008). The common signaling pathways
shared by both the cytokines involve phosphoryla-
tion of STAT6, which leads to dimerization and
translocation of STAT6 to the nucleus where it
Jak1 Jak3 Jak1 Tyk2 ? regulates the transcription of a number of genes.
The type I receptor is predominantly found in cells
STAT6 IRS-2 STAT6
of hemopoietic origin such as lymphocytes and
Grb2 STAT3 (?) granulocytes, resulting from restricted expression
pattern of the IL-2Rg chain. The type II IL-4 recep-
STAT6 STAT6 tor is mainly expressed by cells of nonhemopoietic
PI3K origin such as epithelial cells and fibroblasts.
P P
Some cell types, such as macrophages, express
both receptor types. IL-13 also binds IL-13Ra2,
Hematopoietic cell Nonhematopoietic cell with an extraordinarily high affinity (Lupardus
et al., 2010), which is thought to function as a
STAT6 STAT6 decoy receptor (Chiaramonte et al., 2003), though
PP PP evidence exists for its potential to signal in
selected circumstances (Fichtner-Feigl et al.,
2006).
STAT6
PP
IL-4 triggers proteosomal degradation of both of these phospha- Before global mRNA and protein repertoire analysis, knowl-
tases in bone marrow-cultured mouse macrophages, enhancing edge of AAM markers was restricted to a few molecules.
the PI3K and Akt kinase pathways (Ruschmann et al., 2010). MRC1, MHC class II, FN1, and CCL22 were upregulated in com-
SHIP represses IL-4 production from basophils, thereby indi- mon in both human and mouse macrophages, leading to the
rectly controlling the AAM phenotype (Kuroda et al., 2009). impression that they responded similarly to IL-4 (Gratchev
The mTOR adaptor is an evolutionarily conserved member of et al., 2001; Mantovani et al., 2005; Martinez et al., 2009). Subse-
the PI3K family that forms the core of distinct TORC1 and quent reports showed that important AAM markers in mouse,
TORC2 signaling complexes, broadly responsible for the regula- such as Fizz1, Ym1, and Ym2, lacked homologs in humans,
tion of metabolism, growth, and proliferation (Delgoffe et al., and others such as Arg1 were not upregulated in human macro-
2009). Recent studies have shown that mTOR is essential in phages by IL-4, despite gene homology.
dictating T helper cell differentiation, TORC1 for Th1 and Th17, Microarrays have made it possible to investigate effects of IL-4
and TORC2 for Th2 cell differentiation. It would be interesting in macrophages of both species; however, given the limited
to examine macrophage polarization in mice after genetic abla- number of published studies in human or mouse macrophages,
tion of these distinct pathways. a consensus of markers is hard to establish, particularly for
There are few reports of epigenetic modification or microRNA humans (Syed et al., 2007; Martinez et al., 2006; Scotton et al.,
analysis in AAMs. Signature marker genes of IL-4-treated mouse 2005). Genes that appear to be modulated in common by IL-4
macrophages, Arg1, Ym1, Fizz1, and MRC1, revealed reciprocal in macrophages, monocytes, and perhaps circulating precur-
changes in histone H3K4 and H3K27 methylation; the latter sors include ALOX15, FN1, CD23a, MAO-A, GAS6, FXIIIA1,
methylation markers were removed by the H3K27 demethylase CCL26, CCL22, and IL-17RB. Many of the expected AA markers
Jmjd3 (Ishii et al., 2009). These changes depended on activation could also be regulated by M-CSF, while a proportion of the
of STAT6, which bound to consensus sites at the Jmjd3 pro- genes were uniquely regulated by IL-4.
moter, contributing decreased di- and trimethylation of H3K27, Cell Biology and Membrane Dynamics in AAM
as well as transcriptional activation of AAM marker genes. In Apart from their effects on specific receptors and antigen
vivo studies with Schistosome egg granulomas were consistent markers, IL-4 and IL-13 profoundly alter membrane flow in
with evidence of chromatin remodeling in AAM. macrophages (Kzhyshkowska and Krusell, 2009; Montaner
The kinase Akt regulates LPS-induced microRNA in macro- et al., 1999; Varin and Gordon, 2009). Human blood monocyte-
phages and is implicated in LPS tolerance (Androulidaki et al., derived macrophages displayed enhanced flow from the
2009). The isoforms Akt1 and Akt2 have differential effects on plasma membrane, through early endosomes into an expanded
TLR4 and SOCS1 signaling in macrophages, depending on the vacuolar compartment, accompanied by enhanced fusion with
microRNAs let7e and miR155. The effect of IL-4 and IL-13 on lysosomes (Montaner et al., 1999). These effects are distinct
Akt isoforms has not been reported. Other studies have impli- from those induced by other cytokines, such as IFN-g and
cated microRNAs in regulation of macrophage activation TNF. Apart from receptor-mediated endocytosis, e.g., via man-
(Taganov et al., 2006; Tili et al., 2007). nose, transferrin, and folate receptors (Puig-Kroger et al.,
Review
2009), fluid phase endocytosis is also enhanced. The effects of Ym1 and Ym2 belong to a family of chitinase-like molecules.
IL-4 and IL-13 on phagocytosis have been variable (Varin et al., Chilectins are strongly induced by Th2 cell stimuli on macro-
2009). phages as well as epithelium. Chilectins have undergone consid-
In our hands, with mouse peritoneal macrophages, these cyto- erable diversification during evolution, in many cases losing
kines selectively downregulate phagocytosis of a range of parti- enzyme activity, as with Ym1 and Ym2. These molecules bind
cles, by approximately 50%, independent of specific receptor chitin in insects, Helminth eggs, and Nematode pharynx. Chitin
expression (Varin et al., 2009). Uptake of Neisseria meningitidis induces Th2 cell responses (Reese et al., 2007; Voehringer
via Class A Scavenger receptors, of zymosan particles via Dec- et al., 2006) and can be degraded by glucanases, and MRC1
tin-1, and of latex particles is similarly decreased. Reduced contributes to chitin binding.
uptake is associated with reduced Akt phosphorylation, phago- Fizz1, also known as Relma or Retnla, a secreted protein
cytic cup closure, and PI3 Kinase activation. The mechanism produced by macrophages, epithelium, and eosinophils, is
and functional significance of these observations require further highly induced by IL-13. Fizz1 found in lung allergy and asthma
study. may inhibit inflammation (Nair et al., 2009) Fizz / AAM display
In contrast with reduction of phagocytosis in AAMs, IL-4 is exaggerated antigen-specific Th2 cell differentiation. Fizz1 pro-
required for the efficient uptake of apoptotic neutrophils by tein binds to macrophages and effector CD4+ Th2 cells, inhibit-
macrophages from murine gp91 phox-deficient mice, a model ing Th2 cytokine production via Bruton’s tyrosine kinase. Fizz1
of chronic granulomatous disease (Fernandez-Boyanapalli plays a role in metabolism, as well as regulating T cell differenti-
et al., 2009). ation and eosinophil migration. Others have reported a proinflam-
Another feature of IL-4 and IL-13 effects on macrophages is matory role for Fizz1 and related resistin-like molecules (Munitz
the induction of homokaryon formation by macrophage fusion et al., 2008).
(Helming and Gordon, 2009; Moreno et al., 2007). Foreign
body giant cell formation depends on IL-4, whereas osteoclast Involvement of AAMs in Selected Pathological
formation, induced by RANKL cytokine, is inhibited by IL-4 Processes
(Moreno et al., 2003). Independent studies have demonstrated Role of AAM in Inflammation and Immunity
murine multinucleated giant cell formation by IL-4 and IL-13 AAMs are influenced by, and act upon, many processes associ-
and studied its mechanism. Fusion depends on STAT6 signaling ated with inflammation, immunity, and hypersensitivity. AAMs
and is homotypic, i.e., both fusing partners need to express counteract proinflammatory and cellular immune effector mech-
STAT6 (Moreno et al., 2007). Several surface molecules have anisms, serving regulatory as well as inhibitory functions, to
been implicated in fusion, including E-Cadherin, DC-STAMP shape the nature of the response. For example, IL-4 induces
(dendritic cell-specific transmembrane protein), and CD36, expression of SOCS1 in human and mouse macrophages via
which is thought to interact with patches of exposed PS on STAT6. SOCS1 feedback inhibits IL-4 signaling to limit expres-
fusion partners (Helming and Gordon, 2009). Other molecules sion of STAT6 responsive genes and the AAM pathway (Dicken-
such as DAP-12 induce a fusogenic phenotype required on sheets et al., 2007).
both fusion partners (Helming et al., 2008). The effects of IL-4 Although intimately associated with IL-10, TGF-b, and other
and IL-13 are not necessarily associated with enhanced expres- agents of more profound deactivation, IL-4 and IL-13 promote
sion of fusogenic surface molecules, but may result from clus- a distinctive inflammation and cellular immune response. For
tering in the plasma membrane or altered signaling. example, NO generated by IFN-g-induced iNOS is shut down
With human monocyte-derived macrophages, IL-4 and IL-13 in macrophages (it is induced in epithelial targets), with a shift
accelerate fusion in vitro, but other culture variables, e.g., to Arg1. Interactions with mast cells, basophils, eosinophils,
substratum, growth factors, and serum, also play a role. The and NKT cells, as well as IgE and selected subclasses of IgG,
significance of giant cell formation in granulomatous inflamma- promote allergy and hypersensitivity. Together they influence
tion is obscure, nor is it known whether IL-4 and IL-13 play vascular permeability, angiogenesis, cell recruitment, smooth
a role in macrophage fusion in nonparasitic infections, such as muscle contraction, goblet cell secretion, mucus production,
the prototypical Langhans giant cells of tuberculosis. and collagen deposition by myofibroblasts (Locksley, 2010).
Function of Signature Markers in AAM Several questions remain understudied. For example, the
In the case of enzyme markers such as Arg1, there has been interactions between AAMs and other cells of the innate and
extensive investigation of its possible role in alternative versus acquired immune system, by contact, and secretion, require
classical activation (De’Broski et al., 2010; El Kasmi et al., attention. Do AAMs influence DCs, which themselves are
2008; Herbert et al., 2004; Pesce et al., 2009a). Although its affected by Th2 cytokines, and can AAMs function as antigen-
upregulation in the mouse can be a useful indicator of IL-4 and presenting cells (Loke et al., 2000; Rodriguez-Sosa et al.,
IL-13 effects on macrophages, it can be upregulated by other 2002)? Is the reported upregulation of MHCII significant? Apart
pathways. Its demonstration in human macrophages has been from modulating inflammation, can AAM kill microbial and host
problematic. Its possible role in fibrosis is described below. targets, particularly parasites? Arg1 has been put forward as
Enhanced expression and endocytosis of MRC1 are conserved a candidate. Do IL-4-induced homokaryons participate in this
features in both mouse and human macrophages; enhanced process? Perhaps killing is indirect through eosinophils or
uptake of pathogens through carbohydrate recognition may neutrophils. In sum, the metabolic and secretory activities of
contribute to infection. However, its immunologic role is AAM favor trophic rather than lytic functions, clearance of
unknown. A possible clue is via a role in Th17 cell differentiation apoptotic corpses rather than induction of necrosis, and induc-
(van de Veerdonk et al., 2009). tion of tolerance rather than autoimmunity.
Review
Review
A
Ft IL-4, IL-13
Macrophage Alternatively
activated macrophage Promotes Ft replication
Exogenous
and endogenous IFN-β IL-10
Proinflammatory cytokines
Kills host
Although sterile foreign body reactions can depend on IL-4, properties of AAMs. Polyamines contribute to induction of
IL-4Ra1 chain, and STAT6, repair of surgical wounds was inde- Cadherin-1, after IL-4 treatment in vitro or Taenia crassiceps or
pendent of this pathway (Daley et al., 2010). The IL-13-specific allergen-induced activation in vivo (Van den Bossche et al.,
receptor was not investigated although it has been shown to 2009). Cadherin-1 expression at the macrophage surface,
be essential for fibrosis in a parasitic model of S. mansoni gran- together with catenin, mediates homotypic (cell fusion) and
uloma formation (Ramalingam et al., 2008). As noted, Arg1 can heterotypic interactions with CD103+ T cells. Alternative activa-
be induced by a variety of different pathways, and cells other tion of macrophages has also been implicated in fibrosis after
than macrophages also express receptors for IL-13. The reduc- gamma herpes virus infection in mice (Gangadharan et al., 2008).
tion of fibrosis in egg granulomata in macrophage-selective It is not clear to what extent Th2 cytokines, AAMs, or fibro-
ablation of the IL-13Ra1, in which induction of AAM is unaf- blasts contribute to noninfectious causes of fibrosis or whether
fected, confirms the existence of AAM-independent pathways combined targeting of IL-13 and TGF-b might prevent or reverse
of fibrogenesis, and also independent of TGF-b or MMP9, which fibrosis.
are enhanced in this model. A remarkable example of a pathogenic role of IL-13 in chronic
Macrophage-specific ablation of Arg1 enhances S. mansoni obstructive pulmonary disease (COPD) has been reported
egg-induced fibrosis, an unexpected result. In wild-type mice, (Holtzman et al., 2009; Kim et al., 2008). COPD is a major cause
granulomata appear in liver after 7–10 weeks and diminish of morbidity and mortality, in which lung failure is associated with
subsequently by Arg1-dependent downregulation, which is chronic inflammation, mucous cell metaplasia, bronchial hyper-
absent in Arg1-deficient mice. Macrophages express several reactivity, and fibrosis. The condition is often attributed to aller-
transporters such as Cat2 (Thompson et al., 2008) as well as gens and adaptive immune mechanisms, but this study provides
enzymes involved in L-arginine uptake and metabolism, but convincing evidence that an initial transient acute viral infection
not a complete urea cycle. Ornithine decarboxylase, hydroxy- in the lung can be followed after a period by activation of NKT
proline, and polyamines have been implicated in additional cells, production of IL-13 by these cells, and by macrophages
Review
themselves. The macrophages upregulate IL-13Ra1 expression reduced in macrophages from the hearts of IL-13 or doubly defi-
and become alternatively activated by an autocrine or paracrine cient IL-13 and IL-4, but not IL-4-deficient, mice.
mechanism. PPARs and AAMs: Role in Metabolism, Inflammation,
In an experimental mouse model of Sendai virus infection, and Atherosclerosis
bronchiolitis is associated with mucus production and airway Since the early observation by Glass and colleagues that PPAR
hyperreactivity to methacholine. After the virus becomes barely gamma is strongly induced by IL-4, PPARs have been investi-
detectable, NKT cells expressing semi-invariant Valpha14-J- gated in relation to lipid metabolism, macrophage polarization,
alpha 18 TCR produce IL-13 through a CD1d-glycolipid-depen- obesity and insulin resistance, and atherogenesis (reviewed by
dent mechanism, initiating a vicious circle of AAM amplification. Glass [Glass and Saijo, 2010] and Chawla [Chawla, 2010]).
In the mouse, the characteristic signature markers Arg1, Ym1 Macrophages express PPARg and PPARd; macrophage-spe-
and Ym2, Fizz1, Mmp12, and Alox12e are now present. cific deficient mice have been utilized in bone marrow chimera
An analogous mechanism has been proposed for human experiments of experimental atherosclerosis. Although there
COPD, with patient transplant-derived material to demonstrate has been progress in analyzing the control of inflammation, the
the presence of NKT cells and IL-13 message and protein in therapeutic application of PPAR ligand agonists via these recep-
macrophages (Holtzman et al., 2009). The critical role of IL-13 tors remains elusive. Macrophages in white adipose tissue
in the mouse model was demonstrated by macrophage deple- in obese versus lean mice differ in phenotype, with features of
tion studies and blockade with soluble IL-13R2 protein. This CAMs in the former and of AAMs in the latter. By contrast,
provocative study shifts the pathogenic mechanism to the innate lipid-laden foam cells in atherosclerotic plaques have a distinct
immune system. Improved AAM markers are needed for human phenotype, not necessarily corresponding to the binary classifi-
tissue analysis and prospective studies in human subjects. cation of M1- and M2-polarized cells. AAMs could play a role in
Plasma Chitinase-1, a member of the chitinase family, allows smooth muscle cell migration and plaque rupture.
quantitative stratification of COPD patients (Agapov et al., 2009). IL-4 enhances uptake and oxidation of fatty acids and biogen-
The question arises of genetic susceptibility to these sequelae of esis of mitochondria, via STAT6, regulating programs controlled
common virus infections, as does the possibility of therapeutic by PPARg and the coactivator protein PGC-1b. PPARg is
targeting of the IL-13 pathway. dispensable for induction of AAM but required to sustain it. It
LPS tolerance, a refractory state in responses of the host and plays a more complex role in limiting proinflammatory gene
macrophages to repeated challenge with LPS, is known to expression by LPS, preventing turnover of NCoR corepressor
involve NF-kB. Patients with sepsis can display features resem- complexes and keeping such genes in the ‘‘off’’ position in
bling alternative activation of monocytes. Porta and colleagues AAMs, thus controlling the initiation, magnitude, and duration
have shown that tolerance and alternative activation (M2 polari- of inflammation.
zation) are related processes mediated by p50 homodimers, PPARg regulates primarily the metabolic phenotype of AAMs,
which lack a transcription activation domain of members of the whereas PPARd regulates the immune repertoire. PPARd is
NF-kB pathway (Porta et al., 2009). Studies were performed a sensor of triglyceride VLDL and contributes to macrophage
with human monocyte-derived macrophages and with mouse numbers and phenotype in liver as well as adipose tissue.
primary macrophages, in vitro and in vivo. P50 was shown to Mono-unsaturated fatty acids, such as oleic acid, synergize
be a negative regulator of classical, M1 polarization and of with IL-4 to enhance expression of AAM signature genes; PPARd
IFN-b production, but played a positive, nonredundant role in may also play a role in the proliferative and anti-inflammatory
induction of several signature M2-associated genes. Markers effects of IL-4 on macrophages.
included selected chemokines (CCL2, CCL17, and CCL22),
Arg1, Ym1, and Fizz1. There were some discrepancies between Role of AAMs in Tumorigenesis
human and mouse phenotypes, however, and no reported Macrophages are a prominent component of the stroma and
involvement of IL-4 and IL-13 expression or of their receptors. leukocyte infiltrate in human and experimental mouse tumors
P50 / mice lacked the characteristic AAM phenotype associ- (DeNardo et al., 2009; Gocheva et al., 2010; Grivennikov et al.,
ated with Taenia crassiceps infection, consistent with the overall 2010; Hallam et al., 2009; Joyce and Pollard, 2009; Mantovani
hypothesis. et al., 2008; Sinha et al., 2007). They interact with malignant
In another model, the prototypical proinflammatory function of epithelial, blood vessels and lymphatics, lymphocytes, innate
TLR4 can be subverted by house dust mite allergen, which cells, and extracellular matrix. They have been implicated in
mimics MD2, its coreceptor, to induce IL-4 production (Tromp- carcinogenesis, the angiogenic switch, local invasion, and
ette et al., 2009). metastasis. Systemic features of malignancy have also been
Although IL-13 has been associated with disease-promoting ascribed to macrophages, locally and outside the actual tumor
activities, it can also play a protective role by virtue of its ability bed. It is established that they contribute trophic functions to
to regulate macrophages in an autoimmune myocarditis model the emergence of nascent tumor clones, phagocytose apoptotic
of disease (Cihakova et al., 2008). Infection of mice with Cox- tumor cells, influence the tissue response to hypoxia and,
sackie virus B3 induces a self-limiting myocarditis in most mouse together with regulatory T lymphocytes, suppress Th1 cell and
strains, but a few strains develop severe myocardiopathy, with cytotoxic T lymphocyte antitumor responses.
Th1 cell type inflammation, myosin antibodies, and fibrosis, The nature of tumor-associated macrophages (TAMs) has
especially in IL-13 / , but not IL-4 / , BALB/c mice. T cells are become of great interest, though confusing because of pheno-
not known to express IL-13Ra, and the protective role of IL-13 typic heterogeneity. TAMs have many properties of AAMs;
has been attributed to macrophages. MRC1 expression was myeloid-derived suppressor cells (MDSC) are less defined and
Review
in mouse contain infiltrating GR-1 intermediate CD11b+ mono- ruff et al., 2009). Various strategies have been employed to target
cytes, as well as GR-1 high CD11b+ granulocytic cells (Gabrilo- IL-13 through receptor-specific antibodies, chimeric proteins,
vich and Nagaraj, 2009). Some MDSC express F4/80 in the or soluble receptor (Blease, 2008). Proof-of-concept animal
mouse and IL-4Ra, enabling them to respond to both IL-4 and and early human studies have been undertaken, to ameliorate
IL-13. Innate and adaptive T cells as well as tumor cells can asthma and idiopathic lung fibrosis. Species differences need
produce these cytokines, and some tumors also express the to be borne in mind, because in contrast with the mouse, human
IL-4 and IL-13 alpha receptor (Mandruzzato et al., 2009). decoy IL-13 receptor is not secreted from cells (O’Toole et al.,
Several recent studies with experimental tumors in mice 2008). Novel strategies include functional variants based on
(transplantable as well as oncogene-induced) provide striking analysis of human SNPs that demonstrate altered affinity for
evidence that TAMs have much in common with AAMs. They IL-13Ra2 and cell penetrant proteins or small molecules that
contribute to local invasion through cathepsin B and S, promote target the signaling pathway through STAT 6. Structure-function
angiogenesis and tumor growth through VEGF, and induce studies should aid in the development of IL-13-specific
recruitment of other hemopoietic cells, through chemokines therapeutic agents (LaPorte et al., 2008; Mitchell et al., 2010;
(Gocheva et al., 2010). AAMs as well as tumor cells themselves Lupardus et al., 2010).
orchestrate tumor growth and spread through M-CSF, TNF,
IL-10, TGF-b, and other products. The evidence that alternative Conclusion and Further Questions
activation of macrophages is important in promoting tumorigen- Although there has been striking progress in understanding the
esis includes expression of murine signature markers and mechanisms and functions of AAM, much remains to be discov-
dependence on the IL-4 and IL-13 common receptor alpha ered. We have argued in favor of a restricted definition, shown
chain. Blockade of this receptor or its genetic ablation may the value of in situ analysis in mouse and man, and pointed
therefore shift the host-tumor balance toward tumor rejection to the possibility of selective targeting of pathogenic pathways
by Th1 cell-dependent effector mechanisms, such as IL-12 through IL-13. Major human diseases, including infection,
versus IL-10 production. A recent study (Weiss et al., 2009) inflammation, fibrosis, and malignancy, demonstrate the two-
combined treatment with IL-2 and anti-CD40 to induce a switch edged nature of AAM function at different stages of disease.
from M2 to M1 macrophage functions, depending on CCR2 and The need for validated human markers is apparent. Perhaps
IFN-g. The contribution of TAM to this balance varied in different less obvious is a clearer understanding of what determines the
tumor models and could affect either primary or secondary spectrum of innate, classical, alternative, and other activation
growth. B cell-dependent mechanisms have been demonstrated phenotypes in macrophage populations and individual cells.
in selected tumors (Andreu et al., 2010), with possible interac- The trophic, antigen-presenting, and killing functions of AAMs
tions with AAM Fc receptors. need better characterization. Finally, does alternative activation
IL-13 and its receptors have been implicated in a different of macrophages extend to other innate cells, in particular den-
model of suppression of host immune effector responses to dritic, NKT, and even nonhematopoietic cells? As in other fields
tumors (Fichtner-Feigl et al., 2008a). IL-4 or IL-13, acting through of immunobiology, the alternative may indeed be the antecedent
the IL13Ra1, is able to upregulate expression of the IL-13Ra2 path in evolution.
polypeptide. TNF and NF-kB contribute in parallel to the
STAT6 pathway. IL-13 acts on GR1 intermediate CD11b high ACKNOWLEDGMENTS
monocytic cells, through variant AP1 transcription factor, to
produce TGF-b. This pathway is responsible for suppression of We thank colleagues for sharing unpublished results, Stefanie Vogel and Keri
tumor rejection by Th1 cell-type immune response. Treatment Ann Shirey for Figure 3, and Thiru Ramalingam for Figure 2. This review was
prepared while S.G. was a visiting NCI/NIAID scholar and he thanks Giorgio
with anti-TNF or siRNA to block IL-13Ra2 reduces metastasis Trinchieri and Alan Sher for their hospitality. F.O.M. was supported by the
and promotes survival. British Heart Foundation.
In summary, AAMs are important candidates for therapeutic
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