title: Phenotypic activation and pharmacological outcomes of spontaneously differentiated human monocyte-derived macrophages.
abstract: Macrophage activation has been observed in vivo under physiological and pathological conditions, and may represent an attractive target for pharmacological modulation. This study tested the hypothesis that human blood-derived macrophages generated in vitro in the absence of specific macrophage growth factors respond flexibly to activation stimuli and pharmacological treatment. Monocytes were differentiated to macrophages for 7 days in culture in RPMI 1640 with 10% FCS. The resulting population showed predominance of the M2 over M1 phenotype as measured by flow cytometry and the expression of M1 vs. M2 markers was not mutually exclusive. Activation with LPS/IFN-γ for 48 h significantly increased the fraction of surface CD68-expressing cells, the CD14(+)/CD16(-)/CD68(+) subset and cell-bound TNF-α levels, whereas expression of the CC chemokine receptor (CCR)-2 was unchanged. Expression of the M2 markers CD206, CD163 and CX3CR1 was down-regulated following M1 activation compared with resting and after pre-exposure to M2-triggers. By contrast, alternative activation with IL-4/IL-13 for 48 h did not increase M2 markers, while CD206 up-regulation was observed after 7 days. Both activation signals induced changes in gene expression profiles as shown by Q-PCR. Treatment with 100 nM dexamethasone enhanced the M2 morphotype and CD163 expression while preventing LPS/IFN-γ-induced CD163 down-regulation. After 1-week dexamethasone treatment, virtually all cells acquired a CD163(+)/CD206(+)/CX3CR1(+) M2 phenotype. Therefore, these protocols appear to be useful to perform screens of pharmacological agents targeting human macrophage activation.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Tedesco
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy. Electronic address: chiara.bolego@unipd.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Bolego
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Toniolo
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Nassi
authors.affiliation: Department of Medicine, University Hospital, Italy; Venetian Institute of Molecular Medicine, Padua, Italy.
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Fadini
authors.affiliation: Department of Medical Biotechnologies and Translational Medicine, University of Milan, Italy; Humanitas Clinical and Research Center, Rozzano, Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Locati
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cignarella
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D010641:Phenotype; D018417:Th1 Cells / Q000276:immunology; D058408:Th1-Th2 Balance; D018418:Th2 Cells / Q000276:immunology
chemical_list: D000893:Anti-Inflammatory Agents; D015703:Antigens, CD; D015415:Biomarkers; D016207:Cytokines; D003907:Dexamethasone
keywords: Cytokines; Dexamethasone; Macrophages; Pharmacological targeting; Polarization