Research paper Systematic validation of specific 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 Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands b Department of Med ical Biochemistry, Academic Medical Center/University of Amsterdam, The Netherlands c Department of Experimental Immunology, Academic Medical Center/University of Amsterdam, The Netherlands d Department of Rheumatology, Radboud University Nijmegen Medical Center and Nijmegen Institute for Infection, Inflammation and Immunity, The Netherlands article info abstract Article history: Received 11 July 2011 Received in revised form 17 October 2011 Accepted 18 October 2011 Available online 29 October 2011 Background: Polarization of macrophages by specific micro-environmental conditions impacts upon their function following subsequent activation. This study aimed to systematically vali- date 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 (MΦ IFN-γ ) specifically enhanced membrane expression of CD80 and CD64, IL-4 polarized macrophages (MΦ IL-4 ) mainly upregulated CD200R and CD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MΦ IL-10 ) selective- ly 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 (MΦ GM-CSF ) upregulated CD80 but not CD64 expression, showing a partial phe- notypic similarity with MΦ IFN-γ , and also upregulated the expression of the alternative activa- tion marker CD206. M-CSF polarized macrophages (MΦ M-CSF ) not only expressed increased levels of CD163 and CD16, resembling MΦ IL-10, but also displayed high levels of CD64. The phe- notype of MΦ M-CSF could be further modulated by additional polarization with IFN-γ, IL-4, or IL-10, whereas MΦ GM-CSF showed less phenotypic plasticity. Conclusion: This study validated CD80 as the most robust phenotypic marker for human MΦ IFN-γ , whereas CD200R was upregulated and CD14 was specifically downregulated on MΦ IL-4 . CD163 and CD16 were found to be specific markers for MΦ IL-10 . The GM-CSF/M-CSF differentiation model showed only a partial phenotypic similarity with the IFN-γ/IL-4/IL-10 induced polarization. © 2011 Elsevier B.V. All rights reserved. Keywords: Macrophage polarization Cell surface molecules Phenotypic markers Flow cytometry Inflammation 1. 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 circulat- ing monocytes, these cells differentiate upon entry into tis- sues where they can subsequently be activated by a wide array of microbial and self antigens. A large body of evidence Journal of Immunological Methods 375 (2012) 196–206 Abbreviations: APC, antigen presenting cell;ATM, adipose tissue macro- phage;ERK, extracellular signal-regulated kinase;FIZZ-1, found in inflamma- tory zone-1;GAPDH, glyceraldehyde 3-phosphate dehydrogenase;JNK, Jun N-terminal kinase;MAPK, mitogen-activated protein kinase;PD-L2, pro- grammed death ligand-2;TAM, tumor associated macrophage. ⁎ Corresponding author at: Department of Clinical Immunology and Rheu- matology, 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 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim