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onocytes and tissue macrophages provide both immediate defense against foreign agents and assist during
the setting off and development of the adaptive immune response. Monocytes originally derive from CD34 myeloid
progenitor cells in the bone marrow, circulate in the bloodstream,
and enter peripheral tissues where they mature into different types
of resident macrophages, characterized by low oxygen consumption, low protein synthesis rate, and modest cytokine production
(1, 2). Inflammation due to tissue damage or infection results in
resident macrophage activation, which increases the production of
cytokines, chemokines, and other inflammatory mediators, as well
as monocyte recruitment. In the context of specific immune response, the cytokine milieu compels mononuclear phagocytes to
express specialized and polarized functional properties. Mirroring
the Th1/Th2 nomenclature, many refer to polarized macrophages
as M1 and M2 cells (3 6). Classically polarized activated M1
macrophages have long been known to be induced by IFN- alone
or in concert with microbial stimuli as LPS, or cytokines as TNF
and GM-CSF. M1 cells have an IL-12high, IL-23high, IL-10low phenotype, are proficient producers of effector molecules (reactive ox*Istituto Clinico Humanitas, Rozzano, Italy; Institute of General Pathology, University of Milan, Milan, Italy; and Sir William Dunn School of Pathology, University
of Oxford, Oxford, United Kingdom
ygen and nitrogen intermediates) and inflammatory cytokines (IL1, TNF, IL-6), contribute as inducer and effector cells in
polarized Th1 responses, and mediate resistance against intracellular parasites and tumors (711). In contrast, the alternative M2
form of macrophage activation is a generic name used for various
forms of nonclassically activated macrophages resulting from cell
exposure to IL-4 or IL-13, immune complexes, IL-10, glucocorticoid, or secosteroid (vitamin D3) hormones (3, 9, 12). The various
forms of M2 macrophages share an IL-12low and IL-23low phenotype, generally display high levels of scavenger, mannose (13), and
galactose-type receptors (3), and arginine metabolism is shifted to
production of ornithine and polyamines via arginase (14).
Previous studies have addressed the issue of profiling gene expression in M1 or M2 macrophage activation in the mouse, leading
to the identification of new molecules expressed in polarized murine macrophages (e.g., Ym1, Fizz1, MRC1) (13, 15, 16). Data on
human mononuclear phagocytes on the contrary are scanty and
have highlighted important interspecies differences in key molecules, such as arginase and inducible NO synthase, rendering difficult extrapolation (17, 18). In this study, we report for the first
time a whole genome transcriptional profile analysis of the human
monocyte-to-macrophage differentiation and polarized activation
processes, describing distinct molecular signatures which shed
new light on these processes and reveal new candidate markers.
Received for publication March 9, 2006. Accepted for publication August 3, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Italian Association for Cancer Research, Ministero
dellIstruzione dellUniversita` e della Ricerca (Fondo Investimenti Ricerca di Base,
Progetto di Rilevante Interesse Nazionale, and Consiglio Nazionale delle Ricerche
funding), Fondo Interno per la Ricerca Scientifica e Tecnologica (FIRST Project),
Ministero della Salute, Fondazione Cariplo (NOBEL Project), and the European
Commission (Innochem Project, FP6-518167; Mugen Project, LSHG-CT-2005005203). F.O.M. is a recipient of the International PhD program in Cellular and
Molecular Biology fellowship from Vita-Salute San Raffaele University.
Cell preparation
Address correspondence and reprint requests to Dr. Massimo Locati, Istituto Clinico
Humanitas, Via Manzoni 56, I-20089 Rozzano, Italy. E-mail address: massimo.
locati@humanitas.it
Copyright 2006 by The American Association of Immunologists, Inc.
Human monocytes were obtained from normal blood donor buffy coats by
two-step gradient centrifugation followed by an additional step using the
0022-1767/06/$02.00
Comprehensive analysis of the gene expression profiles associated with human monocyte-to-macrophage differentiation and
polarization toward M1 or M2 phenotypes led to the following main results: 1) M-CSF-driven monocyte-to-macrophage
differentiation is associated with activation of cell cycle genes, substantiating the underestimated proliferation potential of
monocytes. 2) M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic
conditions a default shift toward M2 occurs. 3) Modulation of genes involved in metabolic activities is a prominent feature
of macrophage differentiation and polarization. 4) Lipid metabolism is a main category of modulated transcripts, with
expected up-regulation of cyclo-oxygenase 2 in M1 cells and unexpected cyclo-oxygenase 1 up-regulation in M2 cells. 5) Each
step is characterized by a different repertoire of G protein-coupled receptors, with five nucleotide receptors as novel M2associated genes. 6) The chemokinome of polarized macrophages is profoundly diverse and new differentially expressed
chemokines are reported. Thus, transcriptome profiling reveals novel molecules and signatures associated with human
monocyte-to-macrophage differentiation and polarized activation which may represent candidate targets in pathophysiology. The
Journal of Immunology, 2006, 177: 73037311.
7304
Western blot
After removing the medium, cells were washed in PBS and lysed in icecold lysis buffer (2% Triton X-100, 10 mM Tris-HCl (pH 8), 150 mM
NaCl, 2 mM NaN3, 2 mM EDTA) containing protease inhibitors (Roche
Molecular Biochemicals) for 45 min at 4C. Lysates were harvested and
centrifuged at 13,400 g to eliminate nuclei. Protein concentration was
determined using the bicinchoninic acid assay (Pierce) and 30 g of protein was electrophoresed in a 7.5% SDS-PAGE under nonreducing conditions and transferred to nitrocellulose using standard procedures. PTGS1
and PTGS2 were detected using the specific mAbs CXIII and CX229
(Alexis).
Results
Global transcriptome analysis
The transcriptional events associated with M-CSF-dependent
monocyte-to-macrophage differentiation and subsequent M1 or
M2 cell polarization induced by LPS plus IFN- or IL-4, respectively, were investigated using oligonucleotide microarrays. Results demonstrated the existence of a complex network of gene
regulation and clearly identified specific gene expression patterns
that characterize each phase. PCA analysis was applied to the complete dataset and demonstrated that 98% of the total variance of the
7305
FIGURE 3. GO analysis of human macrophage differentiation and polarization. Hierarchical clustering of the percentage of genes associated
with a GO category with respect to the total number of genes in the cluster.
Columns represent clusters of Fig. 2 (the full graph is present in supplemental figure 7SM).
system lies within the first two components. PCA revealed that
monocyte maturation was associated with a significant modification of the global transcriptome (35% of the total variance), with
larger changes taking place in the early phase of the process
(24% variance in the first 3 days of differentiation), followed by
a smaller overhaul (11% variance in the last 4 days of differentiation) in the late phase (Fig. 1). Macrophage polarization was
also associated with significant changes at the transcriptional level,
although the two polarizing conditions were very different, with
M1 polarization profoundly affecting the transcriptional profile
(90% variance in the shift from M to M1), and M2 polarization
resulting in only subtle adjustments (8% variance in the shift
from M to M2) (Fig. 1).
Differentially expressed genes, selected as described in Materials and Methods, were subjected to figures-of-merit analysis,
where K-means clustering performed optimally for 12 clusters,
with no increase in the predictive value of the algorithm for additional behavioral categories (data not shown). Squared Pearson
correlation was used as similarity measurement, cumulating clusters with mirror performances and generating a total of 6 gene
clusters (Fig. 2). To gain insight into the biological processes involved, each cluster was then subjected to hierarchical subclustering (supplemental figures 1 6)4 and GO analysis as included in the
Expression Analysis Systematic Explorer (26) (Fig. 3).
Monocyte-to-macrophage differentiation was associated with
modulation of 868 (2.2%) transcripts in total (Fig. 2, A and B). Of
4
7306
Gene Name
M1
M2
M1:M2 Ratio
CCR7
IL2RA
IL15RA
IL7R
GPR86
P2RY5
TGFBR2
HRH1
TLR5
DCL-1
MSR1
CXCR4
DECTIN1
P2RY14
DCSIGN
CLECSF13
MS4A6A
CD36
MS4A4A
MRC1
5,893
4,062
2,867
1,553
342
284
218
182
76
179
7
168
130
16
315
93
247
73
16
173
55
181
178
115
1,852
2,610
2,274
1,918
915
2,265
107
2,678
2,520
416
8,355
2,946
8,064
2,713
688
7,343
107
22
16
13
5
9
10
11
12
13
14
16
19
25
27
32
33
37
43
43
4,084
13,338
11,659
15,121
977
6,468
2,402
198
259
2,354
591
316
2,297
1,636
217
270
290
133
19
103
199
260
47
334
164
20
29
343
83
47
446
370
1,212
3,542
5,457
5,028
212
130
59
58
21
19
15
10
9
7
7
7
5
4
6
13
19
38
BCL2A1
FAS
BIRC3
GADD45G
HSXIAPAF1
4,018
2,243
1,318
682
1,928
117
156
162
144
445
34
14
8
5
4
SLC7A5
SLC21A15
SLC2A6
SLC31A2
SLC21A9
SLC4A7
SLC38A6
2,509
371
2,981
4,022
338
43
99
259
52
513
850
2,476
388
2,370
10
7
6
5
7
9
24
INDO
PLA1A
OASL
CHI3L2
HSD11B1
AK3
SPHK1
PFKFB3
PSME2
PFKP
PSMB9
PSMA2
OAS2
CTSC
HEXB
LIPA
16,605
1,854
3,272
1,078
4,066
702
1,974
2,983
10,918
1,618
3,888
4,957
944
3,161
1,582
2,252
142
55
237
89
476
103
324
509
1,789
310
817
1,199
261
13,800
6,809
9,772
117
33
14
12
9
7
6
6
6
5
5
4
4
4
4
4
CXCL11
CCL19
CXCL10
CXCL9
TNF
CCL5
CCL15
IL12B
IL15
TRAIL
IL6
CCL20
PBEF1
ECGF1
IGF1
CCL23
CCL18
CCL13
(Table continues)
Membrane receptors
CCR7
Interleukin 2 receptor chain
Interleukin 15 receptor chain
Interleukin 7 receptor
G protein-coupled receptor 86
Purinergic receptor P2Y5
Transforming growth factor receptor II
Histamine receptor H1
Toll-like receptor 5
C-type lectin receptor DCL-1
Macrophage scavenger receptor 1
CXCR4
C-type lectin superfamily member 12
G protein-coupled receptor 105
CD209
C-type lectin superfamily member 13
Membrane-spanning 4-domains, subfamily A, member 6A
CD36
Membrane-spanning 4-domains, subfamily A, member 4
Mannose receptor C type 1
Cytokines and chemokines
CXCL11
CCL19
CXCL10
CXCL9
Tumor necrosis factor ligand superfamily, member 2
CCL5
CCL15
Interleukin 12B
Interleukin 15
Tumor necrosis factor ligand superfamily, member 10
Interleukin 6
CCL20
Visfatin
Endothelial cell growth factor 1
Insulin-like growth factor 1
CCL23
CCL18
CCL13
Apoptosis-related genes
BCL2-related protein A1
Tumor necrosis factor receptor superfamily, member 6
Baculoviral IAP repeat-containing 3
Growth arrest and DNA-damage-inducible,
XIAP associated factor-1
Solute carriers
Solute carrier family 7, member 5
Solute carrier family 21, member 15
Solute carrier family 2, member 6
Solute carrier family 31, member 2
Solute carrier family 2, member 9
Solute carrier family 4, member 7
Solute carrier family 38, member 6
Enzymes
Indoleamine-pyrrole 2,3 dioxygenase
Phospholipase A1 member A
25-oligoadenylate synthetase-like
Chitinase 3-like 2
Hydroxysteroid (11-) dehydrogenase 1
Adenylate kinase 3
Sphingosine kinase 1
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
Proteasome activator subunit 2
Phosphofructokinase
Proteasome subunit type 9
Proteasome subunit type 2
25-oligoadenylate synthetase 2
Cathepsin C
Hexosaminidase B
Lipase A cholesterol esterase
Gene
Symbol
7307
Table I. (Continued)
Gene Symbol
Adenosine kinase
Histamine N-methyltransferase
Tyrosylprotein sulfotransferase 2
Ceramide kinase
Heparan sulfate 3-O-sulfotransferase 2
Leukotriene A4 hydrolase
Carbonic anhydrase II
Arachidonate 15-lipoxygenase
Heparan sulfate 3-O-sulfotransferase 1
Extracelullar mediators
Pentraxin 3
Chondroitin sulfate proteoglycan 2
Apolipoprotein L3
Insulin-like growth factor binding protein 4
Apolipoprotein L1
Platelet-derived growth factor
Endothelin 1
Apolipoprotein L2
Inhibin A
Apolipoprotein L6
Transforming growth factor -induced protein
Selenoprotein P1
Chimerin 2
Fibronectin 1
Fibrinogen-like 2
DNA-binding factors
Homeobox expressed in ES cells 1
Interferon regulatory factor 1
Activating transcription factor 3
Interferon regulatory factor 7
Growth arrest-specific 7
Early growth response 2
v-maf musculoaponeurotic fibrosarcoma oncogene homolog
ADK
HNMT
TPST2
CERK
HS3ST2
LTA4H
CA2
ALOX15
HS3ST1
M1
M2
M1-M2 Ratio
385
174
132
255
355
380
118
53
42
1,810
935
824
1,431
2,025
2,556
950
600
544
5
5
6
6
6
7
8
11
13
PTX3
CSPG2
APOL3
IGFBP4
APOL1
PDGFA
EDN1
APOL2
INHBA
APOL6
TGFBI
SEPP1
CHN2
FN1
FGL2
914
1,712
4,453
3,821
3,032
422
539
2,027
688
918
1,120
811
76
330
117
30
107
374
349
465
61
86
315
129
202
8,976
7,641
943
8,382
4,569
30
16
12
11
7
7
6
6
5
5
8
9
12
25
39
HESX1
IRF1
ATF3
IRF7
GAS7
EGR2
MAF
1,238
4,288
4,080
2,694
257
108
26
102
527
509
394
1,350
1,238
1,187
12
8
8
7
5
11
46
a
The table shows a selection of genes strictly associated with macrophage polarization grouped into functional categories. M1 and M2 columns report the mean of the
expression values. In each category, genes are ranked according to their fold difference between M1 and M2.
these, 390 (1.0%) genes were transiently regulated during the first
stage of the differentiation process and returned to basal levels in
fully differentiated macrophages (Fig. 2A; list of genes in supplemental figure 1SM). In this cluster, GO analysis highlighted an
overrepresentation of molecules associated with cell cycle (Fig. 3),
including positive modulators of cell proliferation such as cyclins
(A2, that regulates S phase progression; B1 and B2, that regulate
G2-M phase transition; D1 and D3, which regulate G1 phase progression; E2, participating in the late G1-S phase transition) and
cell division-associated proteins 1, 2, 5, 6, 7, and 20 (29 31) (supplemental figure 1SM). Interactome analysis of transiently modulated genes highlighted three main nodes around the genes CDC2,
BCL2L11, and CCL2, as well as the concerted down-regulation of
HLA members. Also notable was the high number of nuclear proteins and the low number of soluble factors among the main regulated genes (Fig. 4A). A second cluster contained 478 (1.2%)
genes rapidly regulated during the differentiation process, maintained in mature macrophages, and essentially refractory to polarizing stimuli (Fig. 2B; list of genes in supplemental figure 2SM).
These transcripts mostly corresponded to membrane receptors, signal transducers, and extracellular proteins functionally related to
the immune response (Fig. 3). Interactome analysis of stably MCSF-regulated genes showed an inversion in the gene distribution,
with a smaller percentage of nuclear genes and an increase of
membrane receptors and soluble factors. It also highlighted three
main nodes around the down-regulated genes IL-1 and IL-8 and the
up-regulated gene apolipoprotein E. A concerted down-regulation
of chemokine receptors, cystatins, and defensins, and an opposite
increase of complement components was also noticed (Fig. 4B).
Gene Name
7308
Discussion
FIGURE 5. Differential regulation of arachidonate metabolism-related enzymes in human macrophage differentiation and polarization. A, Transcriptional analysis reveals a unique regulatory network of the PG synthases
(PTGS), with M1 polarization resulting in increased expression of PTGS2
(COX2) and inhibition of PTGS1 (COX1), and M2 inducing a mirror regulation. B, Western blot analysis confirmed selective induction of COX2 in M1
macrophages and COX1 in M2 macrophages. Results from one experiment are
shown, representative of three independent experiments.
The present study was designed to characterize the gene expression profile of human monocytes undergoing differentiation into
mature macrophages in the presence of M-CSF and subsequent
polarized activation into M1 or M2 cells. These processes were
associated with major changes in the global transcriptome.
Macrophage polarization to M1 was associated with the most
dramatic change in the transcriptome, whereas stimulation with
IL-4 of M-CSF-differentiated macrophages caused a relatively minor alteration in gene expression. This apparently minor effect of
IL-4 is due to the fact that M-CSF-driven differentiation leads per
se to the acquisition of M2 properties, including expression of
mannose receptor 1 and scavenger receptors SR-A. This finding is
in agreement with previous data showing divergent M1-M2 properties of macrophages differentiated in GM-CSF compared with
M-CSF (38, 39). M-CSF is a homeostatic growth factor circulating
at high levels in normal blood. Thus, drifting toward M2 may be
a default pathway in macrophage differentiation.
In particular, monocyte differentiation in the presence of M-CSF
was associated with early (day 3) dramatic regulation of cell-cycle
genes, including the cyclins A2, B1, B2, D1, D3, E2, and CDCA
1, 2, 5, 6, and 7. Though human mononuclear phagocytes, unlike
mouse macrophages, are generally considered terminally differentiated nonproliferating cells, there are reports of human monocytemacrophage proliferation (40, 41). Evidence for proliferation in
GO analysis was used to identify functional categories overrepresented in the panel of genes associated with monocyte differentiation and macrophage polarization. Consistent with the well-recognized capability of macrophages to respond and produce a vast
range of lipidic products, one of the most overrepresented categories was lipid metabolism (Fig. 3). In particular, transcriptional
analysis revealed a unique regulation profile for different enzymes
involved in eicosanoid production (Fig. 5A). Monocyte-to-macrophage maturation was associated with a gradual loss of PG-endoperoxide synthases (both PTGS1 and PTGS2), as well as the
arachidonate 5-lipoxygenase (ALOX5), and leukotriene A4 hydrolase. As expected, classical activation was associated with a
marked induction of cyclooxygenase (COX)-2 (37), accompanied
by a significant unexpected further down-regulation of COX-1, leukotriene A4 hydrolase, thromboxane A synthase 1, and ALOX5.
Conversely, alternative activation resulted in the up-regulation of
7309
FIGURE 6. Repertoire of GPCRs expressed during macrophage differentiation and polarization. A, Hierarchical clustering of differentially expressed GPCRs, obtained using average linkage and Pearson correlation as
distance. B, Real-time PCR confirmation of microarray results. f and
represent M1 and M2 cells, respectively. Statistical analysis was performed
with a two-tailed paired t test in five different blood donors. SE was always
below 5% of the individual means (data not shown). All genes were significantly different in M2 vs control macrophages (p 0.05).
the culture was also confirmed in the present study (data not
shown). Thus, the proliferative potential of human monocytes
should not be underestimated and could be exploited and tailored
for cell expansion.
Modulation of genes involved in general cellular metabolic activities is a prominent feature of macrophage differentiation and
polarization. In addition to providing tools for macrophage function in tissues, these changes may have a more subtle significance.
For instance, macrophages are a major component of adipose tissue and play a role in the metabolic syndrome (42).
Macrophages are an active source of pro- and anti-inflammatory
lipid mediators, such as arachidonic acid derivatives and phosphosphingolipids. COX-2 has long been associated with arachi-
7310
Disclosures
The authors have no financial conflict of interest.
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rate, being still expressed on day 3. LPS and IFN- up-regulate CCR7
and down-regulate CCR1 in mature macrophages, as they do in dendritic cells (48, 49). Presumably, this reciprocal regulation underlies
the trafficking of macrophages to lymph nodes, where their disposal
occurs. Strikingly, of the eight GPCRs highly expressed in the M2
cells, five are nucleotide receptors (Fig. 6), the UDP-glucose receptor
GPR105 being among the most highly regulated genes in human macrophages in response to IL-4. M2 macrophages are associated with
tissue remodeling. High expression of nucleotide receptors endows
M2 cells with sensors for tissue damage (50, 51), and preliminary data
suggest that these ligands modulate relevant functions in this cell type
(data not shown).
The chemokine repertoires of mononuclear phagocytes exposed
to polarizing stimuli are profoundly different (4, 5) and the results
presented here extend this general view. In addition to well-known
polarized chemokines, such as CXCL10 for M1 and CCL17 for
M2 cells, we found high levels of CCL8, CCL15, CCL19, CCL20,
and CXCL13 in M1 cells, and CCL13, CCL14, CCL17, CCL23,
and CCL26 in M2 cells. Association of these molecules with polarized macrophage activation may contribute to pathophysiology.
For instance, we found that M2 cells do not produce detectable
levels of CCL11 but may contribute to the recruitment of CCR3positive leukocytes such as eosinophils, basophils, and some polarized Th2 cells (4) through the expression of CCL26.
A hallmark of M1 polarization is the synthesis of the proinflammatory cytokines IL-6, IL-12, and IL-15 (5) and receptors for
IL-2R -chain, IL-15R -chain, and IL-7R as previously described
in mice (52, 53). In the other pole, M2 are characterized by the
overexpression of several scavenger receptors able to bind a diverse array of endogenous and foreign molecules (54). Our results
confirm the up-regulation by IL-4 of the mannose receptor 1 (13),
the macrophage scavenger receptor 1(36), the C-type lectin-like
receptor Dectin-1 (55) and DC-SIGN (CD209) (56) and report for
the first time in mature macrophages the up-regulation of DCIR,
also called CLECSF6 (57), thoroughly studied in dendritic cells
and the less described C-type lectin DCL-1 (58) and CLECSF13.
Alternatively activated macrophages are also characterized by increased expression of fibronectin (59), which is involved in cell
adhesion and migration processes during embryogenesis, wound
healing, blood coagulation, and metastasis.
The solute carrier family of proteins comprises genes whose
primary role is the transport of divalent cations and small organic
molecules. They regulate transcription through DNA-binding proteins and metal response elements, the activity of enzymes including metalloproteases, superoxide dismutase, inducible NO synthase, and functions like endosomal fusion, and metabolism.
Despite the recognized role of some members in immune disease
susceptibility and infection (60), they have not been associated
with macrophage polarization. We find that classically activated
macrophages are characterized by increased expression of the solute carrier family members SLC21A15 and SLC31A2, while alternatively activated macrophages exhibit increased SLC4A7,
SLC38A6 expression (Table I). The role of these molecules remains to be elucidated.
Hitherto, expression data related to macrophage polarization primarily concern the murine system (3). Investigation of selected
markers in the human system have previously highlighted interspecies discrepancy (17, 61). This report represents the first comprehensive description of the human mononuclear phagocyte system, and provides further evidence of relevant interspecies
variability. For example, IL-4 in this study, as well as IL-13 in our
previous expression-profiling experiments (17), did not induce the
human homolog of the mouse alternative activation markers arginase 1, Fizz1, MMP1 and Ym1. Similarly, a number of molecules
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