Journal of Immunological Methods 375 (2012) 196–206

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Journal of Immunological Methods

j ourna l homepage: www.e lsev ie r .com/ locate / j im

Research paper

Systematic validation of specific phenotypic markers for in vitro polarizedhuman 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 Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlandsb Department of Med ical Biochemistry, Academic Medical Center/University of Amsterdam, The Netherlandsc Department of Experimental Immunology, Academic Medical Center/University of Amsterdam, The Netherlandsd Department of Rheumatology, Radboud University Nijmegen Medical Center and Nijmegen Institute for Infection, Inflammation and Immunity, The Netherlands

a r t i c l e i n f o

Abbreviations: APC, antigen presenting cell;ATM,phage;ERK, extracellular signal-regulated kinase;FIZZ-tory zone-1;GAPDH, glyceraldehyde 3-phosphate deN-terminal kinase;MAPK, mitogen-activated proteigrammed death ligand-2;TAM, tumor associated mac⁎ Corresponding author at: Department of Clinical Im

matology, Academic Medical Center/University of Ams9, 1105 AZ Amsterdam, The Netherlands. Tel.: +31 2

E-mail address: d.l.baeten@amc.uva.nl (D.L.P. Baet

0022-1759/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.jim.2011.10.013

a b s t r a c t

Article history:Received 11 July 2011Received in revised form 17 October 2011Accepted 18 October 2011Available online 29 October 2011

Background: Polarization of macrophages by specific micro-environmental conditions impactsupon their function following subsequent activation. This study aimed to systematically vali-date robust phenotypic markers for in vitro polarized humanmacrophages in order to facilitatethe study of macrophage subsets in vivo.Methods: Human peripheral blood monocytes were polarized in vitro with IFN-γ, IL-4, or IL-10.Similar experiments were performed with TNF, IL-13, dexamethasone, M-CSF and GM-CSF aspolarizing stimuli. Phenotypic markers were assessed by flow cytometry and qPCR.Results: IFN-γ polarized macrophages (MΦIFN-γ) specifically enhanced membrane expressionof CD80 and CD64, IL-4 polarized macrophages (MΦIL-4) mainly upregulated CD200R andCD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MΦIL-10) selective-ly induced CD163, CD16, and CD32. The expression profiles of the most specific markers wereconfirmed by qPCR, dose–response experiments, and the use of alternative polarizing factorsfor each macrophage subset (TNF, IL-13, and dexamethasone, respectively). GM-CSF polarizedmacrophages (MΦGM-CSF) upregulated CD80 but not CD64 expression, showing a partial phe-notypic similarity with MΦIFN-γ, and also upregulated the expression of the alternative activa-tion marker CD206. M-CSF polarized macrophages (MΦM-CSF) not only expressed increasedlevels of CD163 and CD16, resembling MΦIL-10, but also displayed high levels of CD64. The phe-notype of MΦM-CSF could be further modulated by additional polarization with IFN-γ, IL-4, orIL-10, whereas MΦGM-CSF showed less phenotypic plasticity.Conclusion: This study validated CD80 as the most robust phenotypic marker for humanMΦIFN-γ, whereas CD200R was upregulated and CD14 was specifically downregulated onMΦIL-4. CD163 and CD16 were found to be specific markers for MΦIL-10. The GM-CSF/M-CSFdifferentiation model showed only a partial phenotypic similarity with the IFN-γ/IL-4/IL-10induced polarization.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Macrophage polarizationCell surface moleculesPhenotypic markersFlow cytometryInflammation

adipose tissue macro-1, found in inflamma-hydrogenase;JNK, Junn kinase;PD-L2, pro-rophage.munology and Rheu-terdam, Meibergdreef05662895.en).

ll rights reserved.

1. Introduction

Macrophages play a key role in the innate immune systemand drive tissue inflammation in a wide variety of immune-mediated inflammatory diseases. Originating from circulat-ing monocytes, these cells differentiate upon entry into tis-sues where they can subsequently be activated by a widearray of microbial and self antigens. A large body of evidence

197C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

indicates that the macrophage response is not only deter-mined by the type of activation but also heavily depends onthe specific micro-environmental conditions in which cellswere differentiated prior to their activation. The prototypicalexample is activation by TLR ligands such as LPS which,depending on macrophage priming by IFN-γ or immunecomplexes, leads to either pro- or anti-inflammatory cyto-kine production (Anderson and Mosser, 2002a,b; Edwardset al., 2006; Nathan, 1991).

IFN-γ was originally described to polarize macrophagestowards classically activated cells (M1) which secrete highamounts of TNF, IL-12, IL-1β and low amounts of IL-10upon subsequent activation and play an important role infighting intracellular pathogens (Mantovani et al., 2005;Mosser, 2003; Nathan, 1991; O'Shea and Murray, 2008). Incontrast, IL-4 induces alternatively activated macrophages(M2), which are characterized by low pro-inflammatory cy-tokine and high IL-10 production, and are involved in tissuerepair, and anti-parasitic and allergic reactions (Gordon,2003; Gordon and Taylor, 2005; Stein et al., 1992). This polar-ization model has been further refined as factors such asIL-10, glucocorticoids, TGF-β, and immune complexes werealso 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 differencesin cytokine production, the concept of polarization has beenconfirmed by clear differences in chemokine production, NOmetabolism, phagocytosis (Gordon and Taylor, 2005;Mantovani et al., 2004; Martinez et al., 2008, 2009; Mosserand Edwards, 2008) and transcriptional profiles (Ghassabehet al., 2006; Lang et al., 2002; Martinez et al., 2006). Macro-phage polarization is also accompanied by specific changesin cell morphology and phenotype (Gordon and Taylor,2005; Mantovani et al., 2004; Martinez et al., 2008, 2009;Mosser and Edwards, 2008). Already described phenotypicalmarkers are the mannose receptor CD206 and the scavengerreceptor CD163, expression of which is enhanced by IL-4(Chroneos and Shepherd, 1995; Stein et al., 1992) and IL-10, re-spectively (Högger et al., 1998).

The use of subset-specific phenotypic markers may open anew avenue for in vitro functional studies as well as a moreaccurate characterization of the macrophage infiltration in avariety of immune-mediated inflammatory diseases. Howev-er, recent studies have highlighted the complexity and limi-tations of this conceptual model. As a classical example, thetumor associated macrophage (TAM) shares pro- and anti-inflammatory properties and was therefore described as aseparate subset (Chen et al., 2005; Duluc et al., 2007;Mantovani et al., 2002). Another example is the adipose tis-sue macrophage (ATM), which includes M1, M2, and amixed M1/M2 subset, where both phenotype and functiondepend on the local microenvironment and recruited mono-cyte subset (Dalmas et al., 2011; Shaul et al., 2010;Wentworth et al., 2010; Zeyda et al., 2007). These observa-tions raise the crucial question of the exact relationship be-tween phenotype and function in macrophage biology. Onthe other hand, however, the fact that these macrophagetypes cannot be easily classified according to the polarizationmodel may be due to the relative lack of well-validated andspecific phenotypic markers. Firstly, many phenotypic

markers, such as the mouse M2 markers FIZZ-1 and YM-1,have been identified in animal models, but are not expressedon human macrophages (Mantovani et al., 2004). Secondly,for many molecules it still needs to be established whethertheir differential expression at mRNA level truly translatesinto robust differences in protein expression. Thirdly, otherfactors have been proposed to steer polarization besidesIFN-γ, IL-4, or IL-10. Polarization toward M1 versus M2 was,for example, also described to be induced by in vitro expo-sure 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 MΦGM-CSF/MΦIFN-γ and MΦM-CSF/MΦIL-4/MΦIL-10 are phenotypicallysimilar or rather represent distinct cell subsets. Finally, mac-rophages do not necessarily undergo genuine lineage com-mitment as polarization can be reversed both in vitro and invivo (Biswas et al., 2008; Gratchev et al., 2006; Khallou-Laschet et al., 2010; Porcheray et al., 2005; Stout et al.,2005). Therefore, the present study was designed to system-atically validate surface markers for the three main polarizedmacrophage subsets, MΦIFN-γ, MΦIL-4 andMΦIL-10, in humansand to confirm their specificity in different in vitroconditions.

2. Materials and methods

2.1. Monocyte isolation from peripheral blood and in vitropolarization

Monocytes from peripheral blood of healthy volunteerswere isolated by gradient centrifugation with Lymphoprep(Axis-Shield PoPAS, Oslo, Norway) and, subsequently, Percollgradient separation (GE Healthcare, Uppsala, Sweden).Monocytes were cultured at a concentration of 0.5×106/mlin Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen,Breda, The Netherlands) supplemented with 10% fetal calfserum (FCS) (PAA Laboratories, Cölbe, Germany) in 6 wellculture plates (Corning Incorporated, New York, NY, USA)(Van Eijk et al., 2005). Unless indicated otherwise, cellswere polarized with human recombinant IFN-γ (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 Sys-tems) for 4 days. In confirmatory experiments, the cellswere polarized with TNF (50 ng/ml, Biosource, Breda, TheNetherlands), IL-13 (20 ng/ml, Peprotech, London, UK), dexa-methasone (5 nM, Sigma Aldrich, Zwijndrecht, The Nether-lands), M-CSF (50 ng/ml, R&D Systems), or GM-CSF (50 ng/ml, R&D Systems). Finally, in specific experiments, mono-cytes were first differentiated with GM-CSF or M-CSF for4 days and subsequently polarized with IFN-γ, IL-4 or IL-10for another 3 days. Macrophages from different donors werepolarized in independent experiments.

2.2. Flow cytometry

Monocyte-derived macrophages were recovered byscraping of the plate. Surface marker expression was ana-lyzed by flow cytometry on day 4 (BD FACS Calibur Flow Cyt-ometer, Erembodegem, Belgium). Purity was assessed bystaining with anti-CD14 (clone 61D3, eBioscience, SanDiego, CA) and was around 90%. Fluorochrome-labeled

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monoclonal antibodies against CCR2 (clone 48607, R&DSystems, Minneapolis, MN, USA), CCR4 (clone 205410, R&DSystems), CCR7 (clone 2H4, BD Pharmingen, Breda, Nederland),CD1a (clone HI149, BD Pharmingen), CD1c (clone AD5-8E7,Miltenyi biotec, Bergisch Gladbach, Germany), CD11b (cloneM1/70, BD Pharmingen), CD16 (clone DJ130c, AbD Serotec,Düsseldorf, 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, BDPharmingen), CD148 (clone 143–41, R&D Systems), CD163(clone GHI/61, BD Pharmingen), CD180 (clone MHR73, AbDSerotec), CD200R (clone OX108, AbD Serotec), CD206 (clone19.2, BD Pharmingen), CD304 (clone AD5-17F6, Miltenyi bio-tec), HLA-DR (clone G46-6, BD Pharmingen), and gp130(clone 28126, R&D Systems) were used. This non-exhaustivepanel of surface molecules was selected based on reports inmice (Bogdan et al., 1991; Chroneos and Shepherd, 1995;Edwards et al., 2006; Ghassabeh et al., 2006; Gordon andTaylor, 2005; Mantovani et al., 2004; Stein et al., 1992; Stoutet al., 2005), and human cells (Chen et al., 2005; Duluc et al.,2007; Högger et al., 1998; Khallou-Laschet et al., 2010; Koninget 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 moleculesin macrophage activation. The surface expression levels foreach marker were also measured on day 1 and day 7 of polari-zation, after adding fresh IMDM with 10% FCS and polarizingcytokines on day 4. The concentration of the cytokines wasmodulated in dose–response experiments. Donors wereanalyzed in independent experiments and equivalent con-centrations of matched isotype controls were included. Be-fore staining, Fc receptors were blocked with 10% humanserum (Lonza, Cologne, Germany). Data were analyzedwith Flow Jo Flow Cytometry Analysis software (Tree Star,Ashland, OR) after gating on the myeloid population in theFSC/SSC window. Values were expressed as the ratio of thegeometric mean fluorescence intensity (gMFI) of the markerof interest over the gMFI of the isotype control.

2.3. Quantitative real-time PCR

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

Table 1qPCR primer sequences for CD80, CD64, CD200R, CD14, CD163, and CD16. The primDesign (Genscript), as described in Materials and methods.

Forward

CD80 CTGCCTGACCTACTGCTTTGCD64 GCAGGAACACATCCTCTGAACD200R GAGCAATGGCACAGTGACTGTTCD14 AAAGCACTTCCAGAGCCTGTCD163 ACATAGATCATGCATCTGTCATTTCD16 CACCATCACTCAAGGTTTGG

using StepOnePlus™ Real-Time PCR System (Applied Biosys-tems, Foster City, CA). Each 20 μl reaction was performed in a96-well format with 5 ng of cDNA, 10 μl of SYBR green PCRMaster Mix (Applied Biosystems) and a concentration of50 nmol of each primer. All reactions were performed induplicate. The mRNA expression levels were normalizedto those of the human housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH). Oligonucleotideprimerswere designed using the online tool for Real-time PCR (TaqMan)Primer Design (Genscript) and obtained from Invitrogen. Theprimer 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 usedfor comparisons between samples. A P value of less than 0.05was considered to be statistically significant.

3. Results

3.1. Validation of specific phenotypic markers for humanMΦIFN-γ,

MΦIL-4 and MΦIL-10

We investigated the relative expression of a broad panelof surface molecules by flow cytometry after 4 days of invitro polarization of human peripheral blood monocyteswith IFN-γ, IL-4, or IL-10, using unpolarized cells as control(Tabel 2). MΦIFN-γ displayed a robust and specific upregula-tion of the co-stimulatory molecule CD80 and the high affin-ity Fcγ receptor I (CD64), as compared to the unpolarizedand IL-4 or IL-10 polarized macrophages (pb0.001) (Fig. 1Aand B). Compared to all other polarizing conditions, IL-4specifically upregulated the inhibitory receptor CD200R(pb0.001) (Fig. 1D), while it also strongly downregulatedCD14 expression (Fig. 1E) (pb0.05). Finally, MΦIL-10 showeda specific upregulation of the scavenger receptor CD163(Fig. 1G) and the Fcγ receptor III (CD16) (Fig. 1H) versus allother macrophage subsets (pb0.01). The expression of allother investigated surface molecules was not specific forany of the macrophage subsets. In particular, CD86 expres-sion was distinct from CD80, being upregulated by bothIFN-γ and IL-4 (Fig. 1C). The mannose receptor (CD206)was significantly upregulated by IL-4 versus IFN-γ and IL-10(Fig. 1F), but was previously reported to be also induced byGM-CSF and thus not be specific for IL-4 polarizatin(Chroneos and Shepherd, 1995). Fcγ receptor II (CD32) ex-

ers were designed using the online tool for Real-time PCR (TaqMan) Primer

Reverse

GGCGTACACTTTCCCTTCTCGTAACTGGAGGCCAAGCACTGTGGCAGGTCACGGTAGACAATCGTCCAGCTCACAAGGTT

G ATTCTCCTTGGAATCTCACTTCTAAGTCCTGTGTCCACTGCAAA

CD80

15***

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I

Fig. 1. Expression of phenotypic markers on in vitro polarized human macrophages. Monocytes from peripheral blood of healthy donors were cultured for 4 daysin medium or in medium supplemented with IFN-γ, IL-4, or IL-10, and subsequently analyzed by flow cytometry for surface marker expression. The panels depictthe phenotype of the 3 polarized susbets: CD80, CD64, and CD86 expressions for MΦIFN-γ phenotype (panel A to C); CD200R, CD14, and CD206 expressions forMΦIL-4 phenotype (panel D to F); and CD163, CD16, and CD32 expressions for MΦIL-10 phenotype (panel G to I). Bars represent the mean (SEM) of at least 6 in-dependent experiments. *pb0.05, **pb0.01, ***pb0.001.

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pression mimicked the subset-specific expression of CD16, asit was upregulated on MΦIL-10 versus MΦIFN-γ and MΦIL-4

(Fig. 1I), but given the differential regulation of CD32a andCD32b (Supplemental Fig. 1), we excluded this marker fromthe following experiments. Excluding contamination with

Table 2Expression of membrane receptors on unpolarized macrophages, MΦIFN-γ, MΦIL-4, amarker of interest and the gMFI of the isotype control. Values represent the mean±

Medium (fold gMFI) IFN-γ (fold gM

CCR4 2.24±1.08 3.12±0.91CD11b 4.56±0.79 4.25±0.17CD180 1.55±0.30 1.59±0.58CD304 24.88±13.15 16.82±4.08gp130 1.15±0.05 1.40±0.01HLA-DR 33.74±9.03 51.83±33.27

myeloid dendritic cells, both CD1c and CD1a were absent onpolarized macrophages. The plasmacytoid dendritic cellmarker CD304, however, was highly expressed on all macro-phage subsets, as were the myeloid cell marker CD11b andHLA-DR (Table 2). In summary, these data indicate that

nd MΦIL-10. Expression was calculated as the ratio between the gMFI of theSEM.

FI) IL-4 (fold gMFI) IL-10 (fold gMFI)

2.78±1.17 2.47±1.1811.01±1.68 3.90±0.681.70±0.06 2.13±0.52

28.33±7.72 19.14±4.631.14±0.09 1.25±0.12

43.96±15.14 8.58±1.88

200 C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

human MΦIFN-γ specifically express CD80 and CD64, MΦIL-4

upregulate CD200R and downregulate CD14, and MΦIL-10

are characterized by high levels of CD163 and CD16.

3.2. Confirmation of the phenotypic markers for MΦIFN-γ, MΦIL-4

and MΦIL-10

We next aimed to confirm that the candidate markers wererobustly and reproducibly modulated by the polarizing factors.Firstly, we confirmed the induction of these markers at themRNA level by quantitative RT-PCR by comparing the expres-sion levels at 0, 4, and 24 h of exposure to IFN-γ, IL-4, or IL-10.As shown in Supplemental Fig. 2, CD80 and CD64 mRNA levelswere promptly upregulated by IFN-γ, CD200R was inducedand CD14 was inhibited by IL-4, while both CD163 and CD16were increased by IL-10 stimulation. Secondly, we performeda time curve with flow cytometric analysis of the polarizedsubsets at days 0, 4, and 7 to investigate whether the expres-sion of the markers remained stable over time. The surface ex-pression of CD80 and CD64 after IFN-γ polarization and ofCD200R and CD14 after IL-4 polarization was maintained atsimilar levels on days 4 and 7. Following IL-10 polarization,

CD80

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Fig. 2. Time course of candidate markers induction on peripheral blood monocyte-dewas measured by flow cytometry at days 0, 4, and 7 of polarization. Bars represent

CD163 expression was upregulated at day 4 and increasedeven further at day 7. CD16 was already expressed by mono-cytes (day 0) and maintained in time in the presence of IL-10(Fig. 2), but was lost during differentiation with other stimuliin vitro (Fig. 1H). As CD16 expression was previouslyreported on a specific subset rather than all monocytes, wealso assessed the percentage of cells positive for CD16 ratherthan the gMFI; also this analysis showed an increase of CD16on MΦIL-10 compared to the other subsets (data not shown).Thirdly, we repeated the polarization experiments in dose–response conditions for IFN-γ, IL-4, and IL-10 and assessedthe regulation of CD80, CD64, CD200R, CD163 and CD16expression by flow cytometry. All three cytokines induceddose-dependent and specific upregulation of the selectedmarkers (Supplemental Fig. 3). Taken together, these 3 setsof experiments confirmed the specific regulation of the pheno-typic markers of interest by the polarizing stimuli.

3.3. Confirmation of the phenotypic marker specificity for eachpolarized macrophage subset

To examine if the phenotypic markers were specific foreach macrophage subset rather than merely for exposure to

CD64fo

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rived macrophages by IFN-γ, IL-4, or IL-10. Surface expression of the proteinsthe mean (SEM) of 3 independent experiments.

80

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fold gMFI=4.8

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fold gMFI=1.8 fold gMFI=13.8

CD80

CD64fold gMFI=4.8 fold gMFI=5.6fold gMFI=83

fold gMFI=3.8 fold gMFI=25.2 fold gMFI15.1

CD200R

fold gMFI= 1fold gMFI=2.2 fold gMFI=1.2CD14

Medium IFN-γ

Medium IL-4 IL-13

Medium IL-10 Dexamethasone

fold gMFI=2.7 fold gMFI=9.4 fold gMFI=8CD163

fold gMFI=6.7 fold gMFI=41.9 fold gMFI=25.9

CD16

A

B

C

Fig. 3. Expression of the phenotypic markers after in vitro polarization of monocyte-derived macrophages with other subset stimuli. Peripheral blood monocyteswere cultured for 4 days in medium or in medium supplemented with IFN-γ or TNF for CD80 and CD64 expressions (panel A), IL-4 or IL-13 for CD200R and CD14expressions (panel B), and IL-10 or dexamethasone for CD163 and CD16 expressions (panel C). The expression of the surface markers of interest was analyzed byflow cytometry. Data are representative for 3 independent experiments and the histograms represent the gMFI of positively stained cells (black line) compared tothe isotype control (solid gray).

201C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

one particular cytokine, we also measured the expression ofthe proposed phenotypic markers on peripheral blood mono-cytes cultured for 4 days with TNF (which was reported to

polarize macrophages in a similar way as IFN-γ), IL-13 (as asurrogate for IL-4), or with dexamethasone (instead of IL-10, for its immunomodulatory properties) (Ehrchen et al.,

CD64CD80

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Fig. 4. Expression of the phenotypic markers on unpolarized macrophages, MΦGM-CSF, and MΦM-CSF. Peripheral blood-derived monocytes were cultured for 4 daysin 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. *pb0.05, **pb0.01.

202 C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

2007; Högger et al., 1998; Koning et al., 2010; Mantovani etal., 2004; Martinez et al., 2008, 2009; Mosser and Edwards,2008). TNF induced upregulation of CD80 similarly to IFN-γ,but failed to modulate CD64 expression (Fig. 3A). It is impor-tant 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 havethe same effect on polarization as IFN-γ. IL-13 polarizationmodulated CD200R and CD14 expressions comparably to IL-4 (Fig. 3B), while polarization with dexamethasone paral-leled IL-10 in the specific upregulation or maintained ex-pression of CD163 and CD16, respectively (Fig. 3C). Thesedata indicate that the differential expression of the selectedphenotypic markers, with the exception of CD64, reflects

Fig. 5. Expression of the phenotypic markers after polarization of MΦGM-CSF and MΦditional days in medium or medium supplemented with IFN-γ, IL-4, or IL-10. The sBars represent the mean (SEM) of 3 independent experiments. *pb0.05, **pb0.01.

not just the effect of one specific cytokine, but characterizesthe three major polarized macrophage subsets.

3.4. Expression of the phenotypic markers during macrophagedifferentiation with GM-CSF and M-CSF

Besides the previously tested cytokines, GM-CSF andM-CSF were also reported to induce classical versus alternativemacrophage 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 candi-date markers was also specifically modulated by these growthfactors. As shown in Fig. 4, GM-CSF specifically upregulated

M-CSF. GM-CSF or M-CSF differentiated macrophages were cultured for 3 ad-urface expression of the candidate markers was assessed by flow cyotmetry

.

M-CSFGM-CSF

4

5

3

4

5 *

***

CD80

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

Mediu

mIF

N-γIL

-4IL

-10

0

1

2

3

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

fold

gM

FI

40*

****40

0

1

2

CD64

0

10

20

30

0

10

20

30

CD200R

0

10

20

30

40

50

10

20

30

40

50

CD14

0

10

20

30

40

10

20

30

40

CD163 4

6

8

**

**

*

4

6

8

0

0

2

60

40

60

0

2

CD16

0

20

40

0

20

203C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

204 C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

the MΦIFN-γ marker CD80 (pb0.01), but not CD64, comparedto M-CSF and medium control. M-CSF did not significantlymodulate the expression of CD200R and CD14, but upregu-lated the MΦIL-10 markers CD163 (pb0.05) and CD16. As indi-cated previously (Chroneos and Shepherd, 1995), the mouseM2 marker CD206, which was significantly upregulated byIL-4 versus IFN-γ and IL-10, was even stronger upregulatedby GM-CSF in our experiments with human cells, supportingthe notion that CD206 is not a specific marker of IL-4 polariza-tion (Supplemental Fig. 4). Taken together, these data indicatethat there is only a partial phenotypical overlap between theMΦGM-CSF/MΦM-CSF and the MΦIFN-γ/MΦIL-4/MΦIL-10 models.

3.5. Expression of phenotypic markers during polarization ofmature macrophages

In the previous experiments the cells were exposed to po-larizing cytokines during their in vitro maturation frommonocyte to macrophage. Earlier in vitro studies have indi-cated that macrophage polarization can be modified byrenewed exposure to cytokines (Gratchev et al., 2006;Porcheray et al., 2005; Stout et al., 2005). Additionally, it islikely that polarization in vivo is not driven by a single factor,but by simultaneous or sequential exposure to numerouscytokines, or alterations in lipid environment. Therefore, weassessed whether the phenotypic markers reflected this plas-ticity and could be modulated by polarization after initial dif-ferentiation into MΦGM-CSF and MΦM-CSF. Peripheral bloodmonocytes were differentiated for 4 days in the presence ofGM-CSF or M-CSF and were subsequently exposed to IFN-γ,IL-4, or IL-10 for an additional 3 days. As shown in Fig. 5, IFN-γ significantly induced CD80 and CD64 on MΦM-CSF (pb0.05for all comparisons), but not on MΦGM-CSF, while IL-4 induceda slight CD200R upregulation and CD14 downregulation inboth groups. Finally, IL-10 upregulated CD163 and CD16 levelsin both MΦM-CSF and MΦGM-CSF (pb0.05 for MΦGM-CSF, for allcomparisons). Despite the fact that some of these phenotypicalchanges did not reach statistical significance, this experimentshows that the proposed phenotypic markers are specificallyinduced on distinct macrophage subsets not only after primaryin vitro polarization but also – albeit to a lesser extent – aftersecondary polarization of in vitro matured macrophages. Fur-thermore, the phenotypical plasticity of MΦM-CSF seemed tobe higher than that of MΦGM-CSF.

4. Discussion

Macrophages play diverse roles in complex in vivo pro-cesses such as acute and chronic inflammation, tissue repair,and tumor growth. The emerging concept that macrophagefunctions are, at least in part, determined by the polarizationstatus of the cells raised the question whether these distinctpolarized macrophage subsets could be identified by specificphenotypic markers. The main result of the present study isthe validation of CD80 as marker for MΦIFN-γ, CD200R forMΦIL-4, and CD163 and CD16 for MΦIL-10 in humans. CD64appeared to be upregulated only by IFN-γ and not by TNF orGM-CSF, suggesting that its expression may be specific forIFN-γ exposure rather than a universal marker for M1. Fur-thermore, CD16 expression was maintained during in vitromacrophage polarization with IL-10, but not IFN-γ or IL-4.

Four important issues should be consideredwhen interpret-ing these data. Firstly, our results confirm some phenotypicaldifferences between mouse and human macrophages. A strik-ing example is the mannose receptor, which is a marker for al-ternatively polarized macrophages in rodents (Chroneos andShepherd, 1995; Stein et al., 1992), butwasmore potently upre-gulated by GM-CSF than by IL-4 on human macrophages. Also,we confirmed that CD200R is specifically expressed by MΦIL-4

in humans, while a previous report showed that CD200R ex-pression in mice is not dependent upon IL-4 (Koning et al.,2010). Secondly, the differences between the GM-CSF/M-CSFdifferentiation and the IFN-γ/IL-4/IL-10 polarization model inthe expression of certain surface markers, such as CD64 orCD206, highlight the complexity of themacrophage phenotypicpolarization, beyond the simple M1/M2 paradigm. Thirdly, weused a candidate surface molecule approach rather than a sys-tematic mRNA or protein expression analysis and, accordingly,the proposed list of phenotypicmarkers is certainly not exhaus-tive. Identification of additional and potentially even more spe-cific markers remains possible. Finally, themarkers were testedfor their specificity for the major macrophage subsets but werenot tested against other cell types. Obviously, markers such asCD16, CD64, and CD80 are also expressed by other cell typessuch as dendritic cells or activated B lymphocytes. Similarly,CD304 was proposed to be a specific marker for pDC versusmDC but also appears to be highly expressed on all macrophagesubsets (Ji et al., 2009). This implies that themacrophage subsetspecific markers identified in the present study should alwaysbe used in combination with a pan-macrophage marker whenanalyzing mixed cell populations in vitro or ex vivo.

The validation of specific phenotypic markers is highly rele-vant for further investigation of different aspects of humanmac-rophage biology. Firstly, it provides a useful tool to study indetail the regulation of macrophage polarization. For example,we could systematically compare here the effect of GM-CSFandM-CSF versus the prototypical polarizing cytokines, demon-strating that M-CSF induces a MΦIL-10 phenotype but did notmimic the effects of IL-4. The similar effect of different cytokineson macrophage polarization may point towards common tran-scriptional programs (STAT6 phophorylation in the case of IL-4and IL-13, for example) which could determine not only thephenotype but also the function of the distinctmacrophage sub-sets. Additionally, these phenotypic markers form an importanttool to study which factors influence macrophage polarizationin vivo, as we demonstrated previously using synovial fluid ofdifferent types of chronic arthritis (Vandooren et al., 2009).

A second potential application of these phenotypicmarkers is the characterization of differentially polarizedmacrophage subsets during tissue inflammation in vivo, in adisease and/or organ specific manner (Hamilton and Tak,2009). We previously reported an upregulation of CD163during synovitis in spondyloarthritis versus rheumatoid ar-thritis (Baeten et al., 2002, 2004) despite similar numbersof CD68+ cells. The markers validated in the present studywill allow performing additional studies to refine this find-ing and determine whether there is a genuine difference inmacrophage polarization between these two diseases. Simi-lar approaches are broadly applicable to all types oftissue inflammation ranging from inflammatory bowel dis-ease to atherosclerosis. An important issue here is the plas-ticity of macrophage polarization, which was extensively

205C.A. Ambarus et al. / Journal of Immunological Methods 375 (2012) 196–206

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-γ was still able to modify the phenotype ofmacrophages previously polarized by M-CSF. As there arenow reports describing this process also in vivo (Biswas et al.,2008; Khallou-Laschet et al., 2010), the phenotypic markersdescribed here may be of particular interest to study the invivo effect of targeted therapies such as anti-TNF, anti-IL-6, oranti-GM-CSF on tissue macrophages.

The third and most crucial aspect that can be studied withthese markers is to what extent the function of a definedmacrophage subset relates to its phenotype. Some phenotyp-ic markers reported here have an established functional rolein macrophage biology, as demonstrated for CD200R andCD163. Upon binding of CD200 expressed by stromal cells,CD200R inhibits the MAPKs, ERK and JNK signaling pathwaysand may thereby contribute to restrain the pro-inflammatorypotential of macrophages (Copland et al., 2007; Gorczynskiet al., 2010; Jenmalm et al., 2006). Similarly, CD163 has beenreported to prevent tissue inflammation by clearing hemoglo-bin/haptoglobin complexes, inducing the antioxidative en-zyme heme oxygenase-1, as well as by being cleaved to asoluble form which can inhibit T cell activation (Moestrupand Møller, 2004; Schaer et al., 2006; Van Gorp et al., 2010).Whether these and other functions are fixed features of thesepolarized macrophage subsets or heavily dependent uponthe activation status of the cells remains to be investigated.The differential expression of Fcγ receptors on polarizedmacrophage subsets, for example, warrant further investiga-tion of how immune complexes may differentially affect thefunction of these cells (Blom et al., 2003; Sutterwala et al.,1998). Whereas reliable phenotypic markers will facilitatethis type of functional studies in vitro, the major challenge,however, remains to define the phenotype-function correla-tion 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 NetherlandsOrganization for Scientific Research (NWO) and by a grant fromthe Dutch Arthritis Foundation (Reumafonds).

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