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 48h 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 48h 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 100nM 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.

Phenotypic activation and pharmacological outcomes of spontaneously differentiated human monocyte-derived macrophages.

TEDESCO, SERENA;BOLEGO, CHIARA;TONIOLO, ALICE;NASSI, ALBERTO;FADINI, GIAN PAOLO;CIGNARELLA, ANDREA
2015

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 48h 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 48h 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 100nM 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3105902
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