title: Human macrophage polarization in vitro: maturation and activation methods compared.
abstract: Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-β production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo.
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands; Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: d.vogel@vumc.nl.
authors.forename: Daphne Y S
authors.initials: DY
authors.lastname: Vogel
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands; Department of Plastic and Reconstructive Surgery, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Judith E
authors.initials: JE
authors.lastname: Glim
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Andrea W D
authors.initials: AW
authors.lastname: Stavenuiter
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Marjolein
authors.initials: M
authors.lastname: Breur
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Priscilla
authors.initials: P
authors.lastname: Heijnen
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands; Department of Neuroscience and Trauma, Blizard Institute, Barts and the London School of Medicine & Dentistry, Queen Mary University of London, E1 2AT London, United Kingdom.
authors.forename: Sandra
authors.initials: S
authors.lastname: Amor
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Christine D
authors.initials: CD
authors.lastname: Dijkstra
authors.affiliation: Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Robert H J
authors.initials: RH
authors.lastname: Beelen
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology*; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D044967:Serum
chemical_list: D016173:Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: Fibrosis; M1 M2 macrophages; Macrophage activation; Macrophage maturation; Multiple sclerosis; Wound healing