title: Systematic validation of specific phenotypic markers for in vitro polarized human macrophages.
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 (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)) 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 (MΦ(GM-CSF)) upregulated CD80 but not CD64 expression, showing a partial phenotypic similarity with MΦ(IFN-γ), and also upregulated the expression of the alternative activation 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 phenotype 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands.
authors.forename: C A
authors.initials: CA
authors.lastname: Ambarus
authors.forename: S
authors.initials: S
authors.lastname: Krausz
authors.forename: M
authors.initials: M
authors.lastname: van Eijk
authors.forename: J
authors.initials: J
authors.lastname: Hamann
authors.forename: T R D J
authors.initials: TR
authors.lastname: Radstake
authors.forename: K A
authors.initials: KA
authors.lastname: Reedquist
authors.forename: P P
authors.initials: PP
authors.lastname: Tak
authors.forename: D L P
authors.initials: DL
authors.lastname: Baeten
mesh_terms: D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D016764:Cell Polarity / Q000235:genetics / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; D007378:Interleukins; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; D016173:Macrophage Colony-Stimulating Factor; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor