Journal of Immunological Methods 375 (2012) 196206

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Research paper

Systematic validation of specic phenotypic markers for in vitro polarized

human macrophages
C.A. Ambarus a, S. Krausz a, M. van Eijk b, J. Hamann c, T.R.D.J. Radstake d, K.A. Reedquist a, c,
P.P. Tak a, D.L.P. Baeten a,
a
b
c
d

Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands
Department of Med ical Biochemistry, Academic Medical Center/University of Amsterdam, The Netherlands
Department of Experimental Immunology, Academic Medical Center/University of Amsterdam, The Netherlands
Department of Rheumatology, Radboud University Nijmegen Medical Center and Nijmegen Institute for Infection, Inammation and Immunity, The Netherlands

a r t i c l e

i n f o

Article history:
Received 11 July 2011
Received in revised form 17 October 2011
Accepted 18 October 2011
Available online 29 October 2011
Keywords:
Macrophage polarization
Cell surface molecules
Phenotypic markers
Flow cytometry
Inflammation

a b s t r a c t
Background: Polarization of macrophages by specific micro-environmental conditions impacts
upon their function following subsequent activation. This study aimed to systematically validate robust phenotypic markers for in vitro polarized human macrophages in order to facilitate
the study of macrophage subsets in vivo.
Methods: Human peripheral blood monocytes were polarized in vitro with IFN-, IL-4, or IL-10.
Similar experiments were performed with TNF, IL-13, dexamethasone, M-CSF and GM-CSF as
polarizing stimuli. Phenotypic markers were assessed by flow cytometry and qPCR.
Results: IFN- polarized macrophages (MIFN-) specifically enhanced membrane expression
of CD80 and CD64, IL-4 polarized macrophages (MIL-4) mainly upregulated CD200R and
CD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MIL-10) selectively induced CD163, CD16, and CD32. The expression profiles of the most specific markers were
confirmed by qPCR, doseresponse experiments, and the use of alternative polarizing factors
for each macrophage subset (TNF, IL-13, and dexamethasone, respectively). GM-CSF polarized
macrophages (MGM-CSF) upregulated CD80 but not CD64 expression, showing a partial phenotypic similarity with MIFN-, and also upregulated the expression of the alternative activation marker CD206. M-CSF polarized macrophages (MM-CSF) not only expressed increased
levels of CD163 and CD16, resembling MIL-10, but also displayed high levels of CD64. The phenotype of MM-CSF could be further modulated by additional polarization with IFN-, IL-4, or
IL-10, whereas MGM-CSF showed less phenotypic plasticity.
Conclusion: This study validated CD80 as the most robust phenotypic marker for human
MIFN-, whereas CD200R was upregulated and CD14 was specifically downregulated on
MIL-4. CD163 and CD16 were found to be specific markers for MIL-10. The GM-CSF/M-CSF
differentiation model showed only a partial phenotypic similarity with the IFN-/IL-4/IL-10
induced polarization.
2011 Elsevier B.V. All rights reserved.

Abbreviations: APC, antigen presenting cell;ATM, adipose tissue macrophage;ERK, extracellular signal-regulated kinase;FIZZ-1, found in inflammatory zone-1;GAPDH, glyceraldehyde 3-phosphate dehydrogenase;JNK, Jun
N-terminal kinase;MAPK, mitogen-activated protein kinase;PD-L2, programmed death ligand-2;TAM, tumor associated macrophage.
Corresponding author at: Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Meibergdreef
9, 1105 AZ Amsterdam, The Netherlands. Tel.: + 31 205662895.
E-mail address: d.l.baeten@amc.uva.nl (D.L.P. Baeten).
0022-1759/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2011.10.013

1. Introduction
Macrophages play a key role in the innate immune system
and drive tissue inflammation in a wide variety of immunemediated inflammatory diseases. Originating from circulating monocytes, these cells differentiate upon entry into tissues where they can subsequently be activated by a wide
array of microbial and self antigens. A large body of evidence

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

indicates that the macrophage response is not only determined by the type of activation but also heavily depends on
the specific micro-environmental conditions in which cells
were differentiated prior to their activation. The prototypical
example is activation by TLR ligands such as LPS which,
depending on macrophage priming by IFN- or immune
complexes, leads to either pro- or anti-inflammatory cytokine production (Anderson and Mosser, 2002a,b; Edwards
et al., 2006; Nathan, 1991).
IFN- was originally described to polarize macrophages
towards classically activated cells (M1) which secrete high
amounts of TNF, IL-12, IL-1 and low amounts of IL-10
upon subsequent activation and play an important role in
fighting intracellular pathogens (Mantovani et al., 2005;
Mosser, 2003; Nathan, 1991; O'Shea and Murray, 2008). In
contrast, IL-4 induces alternatively activated macrophages
(M2), which are characterized by low pro-inflammatory cytokine and high IL-10 production, and are involved in tissue
repair, and anti-parasitic and allergic reactions (Gordon,
2003; Gordon and Taylor, 2005; Stein et al., 1992). This polarization model has been further refined as factors such as
IL-10, glucocorticoids, TGF-, and immune complexes were
also described to lead to M2 profiles (Anderson and Mosser,
2002a; Bogdan et al., 1991; Goerdt and Orfanos, 1999;
Mantovani et al., 2004; Martinez et al., 2008, 2009;
Schebesch et al., 1997). Besides the mentioned differences
in cytokine production, the concept of polarization has been
confirmed by clear differences in chemokine production, NO
metabolism, phagocytosis (Gordon and Taylor, 2005;
Mantovani et al., 2004; Martinez et al., 2008, 2009; Mosser
and Edwards, 2008) and transcriptional profiles (Ghassabeh
et al., 2006; Lang et al., 2002; Martinez et al., 2006). Macrophage polarization is also accompanied by specific changes
in cell morphology and phenotype (Gordon and Taylor,
2005; Mantovani et al., 2004; Martinez et al., 2008, 2009;
Mosser and Edwards, 2008). Already described phenotypical
markers are the mannose receptor CD206 and the scavenger
receptor CD163, expression of which is enhanced by IL-4
(Chroneos and Shepherd, 1995; Stein et al., 1992) and IL-10, respectively (Hgger et al., 1998).
The use of subset-specific phenotypic markers may open a
new avenue for in vitro functional studies as well as a more
accurate characterization of the macrophage infiltration in a
variety of immune-mediated inflammatory diseases. However, recent studies have highlighted the complexity and limitations of this conceptual model. As a classical example, the
tumor associated macrophage (TAM) shares pro- and antiinflammatory properties and was therefore described as a
separate subset (Chen et al., 2005; Duluc et al., 2007;
Mantovani et al., 2002). Another example is the adipose tissue macrophage (ATM), which includes M1, M2, and a
mixed M1/M2 subset, where both phenotype and function
depend on the local microenvironment and recruited monocyte subset (Dalmas et al., 2011; Shaul et al., 2010;
Wentworth et al., 2010; Zeyda et al., 2007). These observations raise the crucial question of the exact relationship between phenotype and function in macrophage biology. On
the other hand, however, the fact that these macrophage
types cannot be easily classified according to the polarization
model may be due to the relative lack of well-validated and
specific phenotypic markers. Firstly, many phenotypic

197

markers, such as the mouse M2 markers FIZZ-1 and YM-1,

have been identified in animal models, but are not expressed
on human macrophages (Mantovani et al., 2004). Secondly,
for many molecules it still needs to be established whether
their differential expression at mRNA level truly translates
into robust differences in protein expression. Thirdly, other
factors have been proposed to steer polarization besides
IFN-, IL-4, or IL-10. Polarization toward M1 versus M2 was,
for example, also described to be induced by in vitro exposure to GM-CSF or M-CSF, respectively (Fleetwood et al.,
2007, 2009; Sierra-Filardi et al., 2010; Verreck et al., 2004,
2006). It remains largely unknown whether MGM-CSF/
MIFN- and MM-CSF/MIL-4/MIL-10 are phenotypically
similar or rather represent distinct cell subsets. Finally, macrophages do not necessarily undergo genuine lineage commitment as polarization can be reversed both in vitro and in
vivo (Biswas et al., 2008; Gratchev et al., 2006; KhallouLaschet et al., 2010; Porcheray et al., 2005; Stout et al.,
2005). Therefore, the present study was designed to systematically validate surface markers for the three main polarized
macrophage subsets, MIFN-, MIL-4 and MIL-10, in humans
and to confirm their specificity in different in vitro
conditions.
2. Materials and methods
2.1. Monocyte isolation from peripheral blood and in vitro
polarization
Monocytes from peripheral blood of healthy volunteers
were isolated by gradient centrifugation with Lymphoprep
(Axis-Shield PoPAS, Oslo, Norway) and, subsequently, Percoll
gradient separation (GE Healthcare, Uppsala, Sweden).
Monocytes were cultured at a concentration of 0.5 10 6 /ml
in Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen,
Breda, The Netherlands) supplemented with 10% fetal calf
serum (FCS) (PAA Laboratories, Clbe, Germany) in 6 well
culture plates (Corning Incorporated, New York, NY, USA)
(Van Eijk et al., 2005). Unless indicated otherwise, cells
were polarized with human recombinant IFN- (50 ng/ml;
R&D Systems, Abingdon, UK), IL-4 (40 ng/ml; Miltenyi Biotec,
Bergisch Gladbach, Germany), or IL-10 (50 ng/ml; R&D Systems) for 4 days. In confirmatory experiments, the cells
were polarized with TNF (50 ng/ml, Biosource, Breda, The
Netherlands), IL-13 (20 ng/ml, Peprotech, London, UK), dexamethasone (5 nM, Sigma Aldrich, Zwijndrecht, The Netherlands), M-CSF (50 ng/ml, R&D Systems), or GM-CSF (50 ng/
ml, R&D Systems). Finally, in specific experiments, monocytes were first differentiated with GM-CSF or M-CSF for
4 days and subsequently polarized with IFN-, IL-4 or IL-10
for another 3 days. Macrophages from different donors were
polarized in independent experiments.
2.2. Flow cytometry
Monocyte-derived macrophages were recovered by
scraping of the plate. Surface marker expression was analyzed by flow cytometry on day 4 (BD FACS Calibur Flow Cytometer, Erembodegem, Belgium). Purity was assessed by
staining with anti-CD14 (clone 61D3, eBioscience, San
Diego, CA) and was around 90%. Fluorochrome-labeled

198

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

monoclonal antibodies against CCR2 (clone 48607, R&D

Systems, Minneapolis, MN, USA), CCR4 (clone 205410, R&D
Systems), CCR7 (clone 2H4, BD Pharmingen, Breda, Nederland),
CD1a (clone HI149, BD Pharmingen), CD1c (clone AD5-8E7,
Miltenyi biotec, Bergisch Gladbach, Germany), CD11b (clone
M1/70, BD Pharmingen), CD16 (clone DJ130c, AbD Serotec,
Dsseldorf, Germany), CD32 (clone AT10, abcam, Cambridge,
UK), CD64 (clone 10.1, BioLegend, Uithoorn, The Netherlands),
CD80 (clone L307.4, BD Pharmingen), CD86 (clone IT2.2, BD
Pharmingen), CD148 (clone 14341, R&D Systems), CD163
(clone GHI/61, BD Pharmingen), CD180 (clone MHR73, AbD
Serotec), CD200R (clone OX108, AbD Serotec), CD206 (clone
19.2, BD Pharmingen), CD304 (clone AD5-17F6, Miltenyi biotec), HLA-DR (clone G46-6, BD Pharmingen), and gp130
(clone 28126, R&D Systems) were used. This non-exhaustive
panel of surface molecules was selected based on reports in
mice (Bogdan et al., 1991; Chroneos and Shepherd, 1995;
Edwards et al., 2006; Ghassabeh et al., 2006; Gordon and
Taylor, 2005; Mantovani et al., 2004; Stein et al., 1992; Stout
et al., 2005), and human cells (Chen et al., 2005; Duluc et al.,
2007; Hgger et al., 1998; Khallou-Laschet et al., 2010; Koning
et al., 2010; Mantovani et al., 2002; Martinez et al., 2006;
Porcheray et al., 2005; Verreck et al., 2006; Zeyda et al.,
2007), as well as potential involvement of specific molecules
in macrophage activation. The surface expression levels for
each marker were also measured on day 1 and day 7 of polarization, after adding fresh IMDM with 10% FCS and polarizing
cytokines on day 4. The concentration of the cytokines was
modulated in doseresponse experiments. Donors were
analyzed in independent experiments and equivalent concentrations of matched isotype controls were included. Before staining, Fc receptors were blocked with 10% human
serum (Lonza, Cologne, Germany). Data were analyzed
with Flow Jo Flow Cytometry Analysis software (Tree Star,
Ashland, OR) after gating on the myeloid population in the
FSC/SSC window. Values were expressed as the ratio of the
geometric mean fluorescence intensity (gMFI) of the marker
of interest over the gMFI of the isotype control.

2.3. Quantitative real-time PCR

Total RNA was isolated from in vitro polarized macrophages using GenElute Mammalian Total RNA Miniprep
Kit (Sigma-Aldrich, St. Louis, TX) and reverse transcribed
using RevertAid H Minus First Strand cDNA Synthesis Kit
(Fermentas, St. Leon-Rot, Germany). RNA concentration was
determined with the Nanodrop (Nanodrop Technologies,
Wilmington, DE). Quantitative real-time PCR was performed

using StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). Each 20 l reaction was performed in a
96-well format with 5 ng of cDNA, 10 l of SYBR green PCR
Master Mix (Applied Biosystems) and a concentration of
50 nmol of each primer. All reactions were performed in
duplicate. The mRNA expression levels were normalized
to those of the human housekeeping gene glyceraldehyde
3-phosphate dehydrogenase (GAPDH). Oligonucleotide primers
were designed using the online tool for Real-time PCR (TaqMan)
Primer Design (Genscript) and obtained from Invitrogen. The
primer sequences are shown in Table 1.
2.4. Statistics
Statistical analysis was performed using Prism software
(GraphPad, La Jolla, CA). Data were expressed as the mean
SEM and ANOVA, followed by Bonferroni post test were used
for comparisons between samples. A P value of less than 0.05
was considered to be statistically significant.
3. Results
3.1. Validation of specic phenotypic markers for human MIFN-,
MIL-4 and MIL-10
We investigated the relative expression of a broad panel
of surface molecules by flow cytometry after 4 days of in
vitro polarization of human peripheral blood monocytes
with IFN-, IL-4, or IL-10, using unpolarized cells as control
(Tabel 2). MIFN- displayed a robust and specific upregulation of the co-stimulatory molecule CD80 and the high affinity Fc receptor I (CD64), as compared to the unpolarized
and IL-4 or IL-10 polarized macrophages (p b 0.001) (Fig. 1A
and B). Compared to all other polarizing conditions, IL4specifically upregulated the inhibitory receptor CD200R
(p b 0.001) (Fig. 1D), while it also strongly downregulated
CD14 expression (Fig. 1E) (p b 0.05). Finally, MIL-10 showed
a specific upregulation of the scavenger receptor CD163
(Fig. 1G) and the Fc receptor III (CD16) (Fig. 1H) versus all
other macrophage subsets (p b 0.01). The expression of all
other investigated surface molecules was not specific for
any of the macrophage subsets. In particular, CD86 expression was distinct from CD80, being upregulated by both
IFN- and IL-4 (Fig. 1C). The mannose receptor (CD206)
was significantly upregulated by IL-4 versus IFN- and IL-10
(Fig. 1F), but was previously reported to be also induced by
GM-CSF and thus not be specific for IL-4 polarizatin
(Chroneos and Shepherd, 1995). Fc receptor II (CD32) ex-

Table 1
qPCR primer sequences for CD80, CD64, CD200R, CD14, CD163, and CD16. The primers were designed using the online tool for Real-time PCR (TaqMan) Primer
Design (Genscript), as described in Materials and methods.

CD80
CD64
CD200R
CD14
CD163
CD16

Forward

Reverse

CTGCCTGACCTACTGCTTTG
GCAGGAACACATCCTCTGAA
GAGCAATGGCACAGTGACTGTT
AAAGCACTTCCAGAGCCTGT
ACATAGATCATGCATCTGTCATTTG
CACCATCACTCAAGGTTTGG

GGCGTACACTTTCCCTTCTC
GTAACTGGAGGCCAAGCACT
GTGGCAGGTCACGGTAGACA
ATCGTCCAGCTCACAAGGTT
ATTCTCCTTGGAATCTCACTTCTA
AGTCCTGTGTCCACTGCAAA

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

***

80
***

60
40
20

iu
m

10

ed
M

CD200R

10

IL
-

IL
-

-
IF
N

iu
m
M

ed

CD14

CD206
*

***
**

15

20

0
IL

-1

-4

40
20

0
-1

IL

-4

0
-1
IL

-4
IL

N
IF

iu
M

ed

0
-1
IL

-4
IL

-
N
IF

iu
M

***

0
m

**

60

iu

***

ed

IL
**

80

fold gMFI

fold gMFI

ed

CD32

**

***

iu
ed

CD16
**

***

10

0
-1
IL

CD163
**

fold gMFI

-4
IL

N
IF

iu
ed

5
0

-1

IL

IL

-
N
IF

iu
M

ed

-4

0
m

10

IF

10

15

20

10

**

30

fold gMFI

**

40

IF

******

fold gMFI

fold gMFI

50

IL

20

10
IL
-

IL
-

-
IF
N

ed

iu
m

30

10

**
***

**

IL
-

40

***

10

CD86
*

IL
-

***

fold gMFI

***

fold gMFI

fold gMFI

15

CD64

***

CD80

IF
N

199

Fig. 1. Expression of phenotypic markers on in vitro polarized human macrophages. Monocytes from peripheral blood of healthy donors were cultured for 4 days
in medium or in medium supplemented with IFN-, IL-4, or IL-10, and subsequently analyzed by flow cytometry for surface marker expression. The panels depict
the phenotype of the 3 polarized susbets: CD80, CD64, and CD86 expressions for MIFN- phenotype (panel A to C); CD200R, CD14, and CD206 expressions for
MIL-4 phenotype (panel D to F); and CD163, CD16, and CD32 expressions for MIL-10 phenotype (panel G to I). Bars represent the mean (SEM) of at least 6 independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001.

pression mimicked the subset-specific expression of CD16, as

it was upregulated on MIL-10 versus MIFN- and MIL-4
(Fig. 1I), but given the differential regulation of CD32a and
CD32b (Supplemental Fig. 1), we excluded this marker from
the following experiments. Excluding contamination with

myeloid dendritic cells, both CD1c and CD1a were absent on

polarized macrophages. The plasmacytoid dendritic cell
marker CD304, however, was highly expressed on all macrophage subsets, as were the myeloid cell marker CD11b and
HLA-DR (Table 2). In summary, these data indicate that

Table 2
Expression of membrane receptors on unpolarized macrophages, MIFN-, MIL-4, and MIL-10. Expression was calculated as the ratio between the gMFI of the
marker of interest and the gMFI of the isotype control. Values represent the mean SEM.

CCR4
CD11b
CD180
CD304
gp130
HLA-DR

Medium (fold gMFI)

IFN- (fold gMFI)

IL-4 (fold gMFI)

IL-10 (fold gMFI)

2.24 1.08
4.56 0.79
1.55 0.30
24.88 13.15
1.15 0.05
33.74 9.03

3.12 0.91
4.25 0.17
1.59 0.58
16.82 4.08
1.40 0.01
51.83 33.27

2.78 1.17
11.01 1.68
1.70 0.06
28.33 7.72
1.14 0.09
43.96 15.14

2.47 1.18
3.90 0.68
2.13 0.52
19.14 4.63
1.25 0.12
8.58 1.88

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

human MIFN- specifically express CD80 and CD64, MIL-4

upregulate CD200R and downregulate CD14, and MIL-10
are characterized by high levels of CD163 and CD16.

3.2. Conrmation of the phenotypic markers for MIFN-, MIL-4

and MIL-10
We next aimed to confirm that the candidate markers were
robustly and reproducibly modulated by the polarizing factors.
Firstly, we confirmed the induction of these markers at the
mRNA level by quantitative RT-PCR by comparing the expression levels at 0, 4, and 24 h of exposure to IFN-, IL-4, or IL-10.
As shown in Supplemental Fig. 2, CD80 and CD64 mRNA levels
were promptly upregulated by IFN-, CD200R was induced
and CD14 was inhibited by IL-4, while both CD163 and CD16
were increased by IL-10 stimulation. Secondly, we performed
a time curve with flow cytometric analysis of the polarized
subsets at days 0, 4, and 7 to investigate whether the expression of the markers remained stable over time. The surface expression of CD80 and CD64 after IFN- polarization and of
CD200R and CD14 after IL-4 polarization was maintained at
similar levels on days 4 and 7. Following IL-10 polarization,

CD163 expression was upregulated at day 4 and increased

even further at day 7. CD16 was already expressed by monocytes (day 0) and maintained in time in the presence of IL-10
(Fig. 2), but was lost during differentiation with other stimuli
in vitro (Fig. 1H). As CD16 expression was previously
reported on a specific subset rather than all monocytes, we
also assessed the percentage of cells positive for CD16 rather
than the gMFI; also this analysis showed an increase of CD16
on MIL-10 compared to the other subsets (data not shown).
Thirdly, we repeated the polarization experiments in dose
response conditions for IFN-, IL-4, and IL-10 and assessed
the regulation of CD80, CD64, CD200R, CD163 and CD16
expression by flow cytometry. All three cytokines induced
dose-dependent and specific upregulation of the selected
markers (Supplemental Fig. 3). Taken together, these 3 sets
of experiments confirmed the specific regulation of the phenotypic markers of interest by the polarizing stimuli.
3.3. Conrmation of the phenotypic marker specicity for each
polarized macrophage subset
To examine if the phenotypic markers were specific for
each macrophage subset rather than merely for exposure to

CD64
80

60

fold gMFI

6
4
2

20

25

30

20

20
10
0

15
10
5

CD163

CD16
40

fold gMFI

6
4
2
0

30
20
10

7d

7d

4d

0d

0d

fold gMFI

7d

0d

7d

4d

0d

10

IL-10

7d

CD14

40

fold gMFI

IL-4

fold gMFI

CD200R

4d

0d

7d

4d

0d

40

4d

IFN-

fold gMFI

CD80
10

4d

200

Fig. 2. Time course of candidate markers induction on peripheral blood monocyte-derived macrophages by IFN-, IL-4, or IL-10. Surface expression of the proteins
was measured by flow cytometry at days 0, 4, and 7 of polarization. Bars represent the mean (SEM) of 3 independent experiments.

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

Medium

CD80

100

80

80

80

60

60

60

20

fold gMFI=1.8

0
100 101 102 103 104

40
20
0

100 101 102 103 104

100
80

60

60

60

40

40

fold gMFI=4.8

fold gMFI=83

40

100 101 102 103 104

0
100 101 102 103 104

IL-4

IL-13

100

100

100

80

80

80

60

60

60

40

40

20

fold gMFI=3.8

0
100 101 102 103 104

20

40

fold gMFI=25.2

0
100 101 102 103 104

20

100

100

80

80

80

60

60

60

40

40

fold gMFI=2.2

0
100 101 102 103 104

Medium

40

IL-10

0
100 101 102 103 104

Dexamethasone

100

100

100

80

80

80

60

60

60

40

40

20

fold gMFI=2.7

0
100 101 102 103 104

20

fold gMFI=9.4

0
100 101 102 103 104

40
20

100

100

80

80

80

60

60

60

20

fold gMFI=6.7

0
100 101 102 103 104

40
20

fold gMFI=8

0
100 101 102 103 104

100

40

fold gMFI=1.2

20

20

0
100 101 102 103 104

fold gMFI= 1

fold gMFI15.1

0
100 101 102 103 104

100

20

fold gMFI=5.6

20

20
0

fold gMFI=4.8

0
100 101 102 103 104

80

Medium

CD16

20

100

CD163

40

80

0
100 101 102 103 104

CD14

fold gMFI=13.8

100

20

CD200R

TNF
100

100

40

CD64

IFN-

201

fold gMFI=41.9

0
100 101 102 103 104

40
20

fold gMFI=25.9

0
100 101 102 103 104

Fig. 3. Expression of the phenotypic markers after in vitro polarization of monocyte-derived macrophages with other subset stimuli. Peripheral blood monocytes
were cultured for 4 days in medium or in medium supplemented with IFN- or TNF for CD80 and CD64 expressions (panel A), IL-4 or IL-13 for CD200R and CD14
expressions (panel B), and IL-10 or dexamethasone for CD163 and CD16 expressions (panel C). The expression of the surface markers of interest was analyzed by
flow cytometry. Data are representative for 3 independent experiments and the histograms represent the gMFI of positively stained cells (black line) compared to
the isotype control (solid gray).

one particular cytokine, we also measured the expression of

the proposed phenotypic markers on peripheral blood monocytes cultured for 4 days with TNF (which was reported to

polarize macrophages in a similar way as IFN-), IL-13 (as a

surrogate for IL-4), or with dexamethasone (instead of IL10, for its immunomodulatory properties) (Ehrchen et al.,

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

CD80

CD64
10

**

fold gMFI

6
4
2

4
2

fold gMFI

6
4
2

SF
-C

-C
M

ed

SF

m
iu

SF
-C

SF
M

-C

iu
ed
M

CD163

CD16

*
fold gMFI

2
1
0

4
2

SF
-C

SF
-C
M
G

ed

iu

SF
-C
M

-C
M
G

ed

iu

SF

fold gMFI

SF

10

0
m

-C

CD14

15

-C
M

ed
M

SF

iu
m

SF
-C

SF
-C
M

ed
M

CD200R

10

fold gMFI

0
iu
m

fold gMFI

10

202

Fig. 4. Expression of the phenotypic markers on unpolarized macrophages, MGM-CSF, and MM-CSF. Peripheral blood-derived monocytes were cultured for 4 days
in medium or in medium supplemented with M-CSF or GM-CSF and were subsequently analyzed by flow cytometry for surface expressions of CD80, CD64,
CD200R, CD14, CD163, and CD16. Bars represent the mean (SEM) of at least 6 independent experiments. *p b 0.05, **p b 0.01.

2007; Hgger et al., 1998; Koning et al., 2010; Mantovani et

al., 2004; Martinez et al., 2008, 2009; Mosser and Edwards,
2008). TNF induced upregulation of CD80 similarly to IFN-,
but failed to modulate CD64 expression (Fig. 3A). It is important to note that TNF was proposed by some (Dalmas et al.,
2011; Gratchev et al., 2001), but not by all authors
(Martinez et al., 2009; Mosser and Edwards, 2008) to have
the same effect on polarization as IFN-. IL-13 polarization
modulated CD200R and CD14 expressions comparably to IL4 (Fig. 3B), while polarization with dexamethasone paralleled IL-10 in the specific upregulation or maintained expression of CD163 and CD16, respectively (Fig. 3C). These
data indicate that the differential expression of the selected
phenotypic markers, with the exception of CD64, reflects

not just the effect of one specific cytokine, but characterizes

the three major polarized macrophage subsets.

3.4. Expression of the phenotypic markers during macrophage

differentiation with GM-CSF and M-CSF
Besides the previously tested cytokines, GM-CSF and
M-CSF were also reported to induce classical versus alternative
macrophage polarization, respectively (Fleetwood et al., 2007,
2009; Sierra-Filardi et al., 2010; Verreck et al., 2004, 2006).
Therefore, we assessed whether the expression of the candidate markers was also specifically modulated by these growth
factors. As shown in Fig. 4, GM-CSF specifically upregulated

Fig. 5. Expression of the phenotypic markers after polarization of MGM-CSF and MM-CSF. GM-CSF or M-CSF differentiated macrophages were cultured for 3 additional days in medium or medium supplemented with IFN-, IL-4, or IL-10. The surface expression of the candidate markers was assessed by flow cyotmetry.
Bars represent the mean (SEM) of 3 independent experiments. *p b 0.05, **p b 0.01.

60

-1

IL

-1

20
0

-1

IL

-4

IL

-1

IL

-4

IL

IF

-1
0

IL

-4

IL

IF

IL
-

10

-
IL
-

IF
N

iu
m

IL

-4

**

IL

**

**

-4

**

ed

fold gMFI

GM-CSF

IL

0
-

30

40

30

IF

40

50

40

IF

40

40

10
m

10

IF

20
m

10

20

30
iu

ed

10

iu

ed

20

iu

30

fold gMFI

IL
-

8
ed

50

fold gMFI

-1
0

IL

-
IL
-

IF
N

iu
m

ed

iu

M
ed

40

fold gMFI

-1

IL

-4

IL

IF

iu

ed

iu

fold gMFI

-1

IL

-4

IL

IF

iu

ed

fold gMFI
4

ed

40

fold gMFI

-1

IL

-4

IL

IF

iu

ed

fold gMFI

-1

-4

IL

IL

-4

IF

iu

ed

IL

N
-

IF

iu

CD16

ed

CD163

CD14
fold gMFI

CD200R

fold gMFI

CD64

fold gMFI

CD80

fold gMFI

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

203

M-CSF
*
*

**
*

30

20

10
0

30

20

0
10

20

10

60

40

20

204

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

the MIFN- marker CD80 (pb 0.01), but not CD64, compared
to M-CSF and medium control. M-CSF did not significantly
modulate the expression of CD200R and CD14, but upregulated the MIL-10 markers CD163 (pb 0.05) and CD16. As indicated previously (Chroneos and Shepherd, 1995), the mouse
M2 marker CD206, which was significantly upregulated by
IL-4 versus IFN- and IL-10, was even stronger upregulated
by GM-CSF in our experiments with human cells, supporting
the notion that CD206 is not a specific marker of IL-4 polarization (Supplemental Fig. 4). Taken together, these data indicate
that there is only a partial phenotypical overlap between the
MGM-CSF/MM-CSF and the MIFN-/MIL-4/MIL-10 models.
3.5. Expression of phenotypic markers during polarization of
mature macrophages
In the previous experiments the cells were exposed to polarizing cytokines during their in vitro maturation from
monocyte to macrophage. Earlier in vitro studies have indicated that macrophage polarization can be modified by
renewed exposure to cytokines (Gratchev et al., 2006;
Porcheray et al., 2005; Stout et al., 2005). Additionally, it is
likely that polarization in vivo is not driven by a single factor,
but by simultaneous or sequential exposure to numerous
cytokines, or alterations in lipid environment. Therefore, we
assessed whether the phenotypic markers reflected this plasticity and could be modulated by polarization after initial differentiation into MGM-CSF and MM-CSF. Peripheral blood
monocytes were differentiated for 4 days in the presence of
GM-CSF or M-CSF and were subsequently exposed to IFN-,
IL-4, or IL-10 for an additional 3 days. As shown in Fig. 5, IFN significantly induced CD80 and CD64 on MM-CSF (pb 0.05
for all comparisons), but not on MGM-CSF, while IL-4 induced
a slight CD200R upregulation and CD14 downregulation in
both groups. Finally, IL-10 upregulated CD163 and CD16 levels
in both MM-CSF and MGM-CSF (pb 0.05 for MGM-CSF, for all
comparisons). Despite the fact that some of these phenotypical
changes did not reach statistical significance, this experiment
shows that the proposed phenotypic markers are specifically
induced on distinct macrophage subsets not only after primary
in vitro polarization but also albeit to a lesser extent after
secondary polarization of in vitro matured macrophages. Furthermore, the phenotypical plasticity of MM-CSF seemed to
be higher than that of MGM-CSF.
4. Discussion
Macrophages play diverse roles in complex in vivo processes such as acute and chronic inflammation, tissue repair,
and tumor growth. The emerging concept that macrophage
functions are, at least in part, determined by the polarization
status of the cells raised the question whether these distinct
polarized macrophage subsets could be identified by specific
phenotypic markers. The main result of the present study is
the validation of CD80 as marker for MIFN-, CD200R for
MIL-4, and CD163 and CD16 for MIL-10 in humans. CD64
appeared to be upregulated only by IFN- and not by TNF or
GM-CSF, suggesting that its expression may be specific for
IFN- exposure rather than a universal marker for M1. Furthermore, CD16 expression was maintained during in vitro
macrophage polarization with IL-10, but not IFN- or IL-4.

Four important issues should be considered when interpreting these data. Firstly, our results confirm some phenotypical
differences between mouse and human macrophages. A striking example is the mannose receptor, which is a marker for alternatively polarized macrophages in rodents (Chroneos and
Shepherd, 1995; Stein et al., 1992), but was more potently upregulated by GM-CSF than by IL-4 on human macrophages. Also,
we confirmed that CD200R is specifically expressed by MIL-4
in humans, while a previous report showed that CD200R expression in mice is not dependent upon IL-4 (Koning et al.,
2010). Secondly, the differences between the GM-CSF/M-CSF
differentiation and the IFN-/IL-4/IL-10 polarization model in
the expression of certain surface markers, such as CD64 or
CD206, highlight the complexity of the macrophage phenotypic
polarization, beyond the simple M1/M2 paradigm. Thirdly, we
used a candidate surface molecule approach rather than a systematic mRNA or protein expression analysis and, accordingly,
the proposed list of phenotypic markers is certainly not exhaustive. Identification of additional and potentially even more specific markers remains possible. Finally, the markers were tested
for their specificity for the major macrophage subsets but were
not tested against other cell types. Obviously, markers such as
CD16, CD64, and CD80 are also expressed by other cell types
such as dendritic cells or activated B lymphocytes. Similarly,
CD304 was proposed to be a specific marker for pDC versus
mDC but also appears to be highly expressed on all macrophage
subsets (Ji et al., 2009). This implies that the macrophage subset
specific markers identified in the present study should always
be used in combination with a pan-macrophage marker when
analyzing mixed cell populations in vitro or ex vivo.
The validation of specific phenotypic markers is highly relevant for further investigation of different aspects of human macrophage biology. Firstly, it provides a useful tool to study in
detail the regulation of macrophage polarization. For example,
we could systematically compare here the effect of GM-CSF
and M-CSF versus the prototypical polarizing cytokines, demonstrating that M-CSF induces a MIL-10 phenotype but did not
mimic the effects of IL-4. The similar effect of different cytokines
on macrophage polarization may point towards common transcriptional programs (STAT6 phophorylation in the case of IL-4
and IL-13, for example) which could determine not only the
phenotype but also the function of the distinct macrophage subsets. Additionally, these phenotypic markers form an important
tool to study which factors influence macrophage polarization
in vivo, as we demonstrated previously using synovial fluid of
different types of chronic arthritis (Vandooren et al., 2009).
A second potential application of these phenotypic
markers is the characterization of differentially polarized
macrophage subsets during tissue inflammation in vivo, in a
disease and/or organ specific manner (Hamilton and Tak,
2009). We previously reported an upregulation of CD163
during synovitis in spondyloarthritis versus rheumatoid arthritis (Baeten et al., 2002, 2004) despite similar numbers
of CD68+ cells. The markers validated in the present study
will allow performing additional studies to refine this finding and determine whether there is a genuine difference in
macrophage polarization between these two diseases. Similar approaches are broadly applicable to all types of
tissue inflammation ranging from inflammatory bowel disease to atherosclerosis. An important issue here is the plasticity of macrophage polarization, which was extensively

C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196206

demonstrated in vitro (Gratchev et al., 2006; Porcheray et al.,

2005; Stout et al., 2005) and confirmed here by the fact that,
for example, IFN- was still able to modify the phenotype of
macrophages previously polarized by M-CSF. As there are
now reports describing this process also in vivo (Biswas et al.,
2008; Khallou-Laschet et al., 2010), the phenotypic markers
described here may be of particular interest to study the in
vivo effect of targeted therapies such as anti-TNF, anti-IL-6, or
anti-GM-CSF on tissue macrophages.
The third and most crucial aspect that can be studied with
these markers is to what extent the function of a defined
macrophage subset relates to its phenotype. Some phenotypic markers reported here have an established functional role
in macrophage biology, as demonstrated for CD200R and
CD163. Upon binding of CD200 expressed by stromal cells,
CD200R inhibits the MAPKs, ERK and JNK signaling pathways
and may thereby contribute to restrain the pro-inflammatory
potential of macrophages (Copland et al., 2007; Gorczynski
et al., 2010; Jenmalm et al., 2006). Similarly, CD163 has been
reported to prevent tissue inflammation by clearing hemoglobin/haptoglobin complexes, inducing the antioxidative enzyme heme oxygenase-1, as well as by being cleaved to a
soluble form which can inhibit T cell activation (Moestrup
and Mller, 2004; Schaer et al., 2006; Van Gorp et al., 2010).
Whether these and other functions are fixed features of these
polarized macrophage subsets or heavily dependent upon
the activation status of the cells remains to be investigated.
The differential expression of Fc receptors on polarized
macrophage subsets, for example, warrant further investigation of how immune complexes may differentially affect the
function of these cells (Blom et al., 2003; Sutterwala et al.,
1998). Whereas reliable phenotypic markers will facilitate
this type of functional studies in vitro, the major challenge,
however, remains to define the phenotype-function correlation in human physiologic and pathologic conditions in vivo.
Authorship
Study design: D.L.P. Baeten, C.A. Ambarus, J. Hamann, K.A.
Reedquist.
Data acquirement: C.A. Ambarus, S. Krausz.
Data analysis: C.A. Ambarus, S. Krausz, M. van Eijk.
Manuscript preparation: C.A. Ambarus, D.L.P. Baeten.
Critical review: S. Krausz, M. van Eijk, J. Hamann, K.A.
Reedquist, T.R.D.J Radstake, P.P. Tak, D.L.P. Baeten.
Manuscript approval: S. Krausz, M. van Eijk, J. Hamann, K.A.
Reedquist, T.R.D.J Radstake, P.P. Tak, D.L.P. Baeten.
Supplementary materials related to this article can be
found online at doi:10.1016/j.jim.2011.10.013.
Acknowledgments
D. Baeten is supported by a VIDI grant from The Netherlands
Organization for Scientific Research (NWO) and by a grant from
the Dutch Arthritis Foundation (Reumafonds).
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