Research paper
Systematic validation of specific phenotypic markers for in vitro polarized human macrophages

https://doi.org/10.1016/j.jim.2011.10.013Get rights and content

Abstract

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-γ, 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-γ polarized macrophages (MΦIFN-γ) specifically enhanced membrane expression of CD80 and CD64, IL-4 polarized macrophages (MΦIL-4) mainly upregulated CD200R and CD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MΦIL-10) selectively induced CD163, CD16, and CD32. The expression profiles of the most specific markers were confirmed by qPCR, dose–response experiments, and the use of alternative polarizing factors for each macrophage subset (TNF, IL-13, and dexamethasone, respectively). GM-CSF polarized macrophages (MΦGM-CSF) upregulated CD80 but not CD64 expression, showing a partial phenotypic similarity with MΦIFN-γ, and also upregulated the expression of the alternative activation marker CD206. M-CSF polarized macrophages (MΦM-CSF) not only expressed increased levels of CD163 and CD16, resembling MΦIL-10, but also displayed high levels of CD64. The phenotype of MΦM-CSF could be further modulated by additional polarization with IFN-γ, IL-4, or IL-10, whereas MΦGM-CSF showed less phenotypic plasticity.

Conclusion

This study validated CD80 as the most robust phenotypic marker for human MΦIFN-γ, whereas CD200R was upregulated and CD14 was specifically downregulated on MΦIL-4. CD163 and CD16 were found to be specific markers for MΦIL-10. The GM-CSF/M-CSF differentiation model showed only a partial phenotypic similarity with the IFN-γ/IL-4/IL-10 induced polarization.

Introduction

Macrophages play a key role in the innate immune system and drive tissue inflammation in a wide variety of immune-mediated 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 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-γ or immune complexes, leads to either pro- or anti-inflammatory cytokine production (Anderson and Mosser, 2002a, Anderson and Mosser, 2002b, Edwards et al., 2006, Nathan, 1991).

IFN-γ was originally described to polarize macrophages towards classically activated cells (M1) which secrete high amounts of TNF, IL-12, IL-1β 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-β, 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, Martinez et al., 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, Martinez et al., 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, Martinez et al., 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 (Högger 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 anti-inflammatory 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 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-γ, 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, Fleetwood et al., 2009, Sierra-Filardi et al., 2010, Verreck et al., 2004, Verreck et al., 2006). It remains largely unknown whether MΦGM-CSF/MΦIFN-γ and MΦM-CSF/MΦIL-4/MΦIL-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, Khallou-Laschet 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, MΦIFN-γ, MΦIL-4 and MΦIL-10, in humans and to confirm their specificity in different in vitro conditions.

Section snippets

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 × 106 /ml in Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen, Breda, The Netherlands) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Cölbe, Germany) in 6 well culture plates (Corning Incorporated, New

Validation of specific phenotypic markers for human MΦIFN-γ,IL-4 and MΦIL-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-γ, IL-4, or IL-10, using unpolarized cells as control (Tabel 2). MΦIFN-γ displayed a robust and specific upregulation of the co-stimulatory molecule CD80 and the high affinity Fcγ receptor I (CD64), as compared to the unpolarized and IL-4 or IL-10 polarized macrophages (p < 0.001) (Fig. 1A and B). Compared to all other

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 MΦIFN-γ, CD200R for MΦIL-4, and CD163 and

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.

The following are the supplementary materials related to

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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