In response to various signals, macrophages undergo classical M1 activation (stimulated by toll-like receptor ligands and IFN-γ) or alternative M2 activation (stimulated by IL-4/IL-13). Functional polarization of macrophages has been observed in vivo under physiological and pathological conditions, and may represent an attractive target for pharmacological modulation. Given the lack of gold standards for M1/M2 activation in vitro, we polarized human monocyte-derived macrophages using different protocols and performed extensive characterization including the modulation of specific markers by dexamethasone. Human monocytes were obtained from healthy donor buffy coats by 2-step gradient centrifugation, and macrophages were obtained by culturing monocytes for 7 days in RPMI 1640 medium with 10% FCS. While virtually 100% of macrophages specifically stained for the intracellular glycoprotein CD68 on immunocytochemistry, only 10% showed surface expression as measured by flow cytometry, indicating a low basal activation state. When resting macrophages were incubated with LPS/IFN-γ (M1) for 4 h, no change was observed in the percentage of cells expressing the M1 surface markers CD68 and CCR2. After longer stimulation (48 h), the number of CD68- but not CCR2-positive cells doubled as compared with resting macrophages. This polarization protocol concomitantly down-regulated the expression of M2 markers CD206, CD163 and CX3CR1. In contrast, none of these markers were affected by alternative (M2) polarization with IL-4/IL-13 for 48 h whereas CD206 was up-regulated after 7 days. Q-PCR phenotyping after M1 polarization for 6-48 h showed increased TNF-α, IL-1β, COX-2, IL-10 and VEGF transcript levels with decreased CD206 and COX-1 expression. Finally, overnight pretreatment with 10 nM dexamethasone enhanced surface expression of CD163 in resting and M2-polarized macrophages, and reversed M1-induced CD163 down-regulation. We conclude that the phenotypic characterization of polarized macrophages is required and provides a basis for pharmacological macrophage targeting.

Phenotypes of human macrophage polarized activation: role of dexamethasone

TONIOLO, ALICE;TEDESCO, SERENA;CIGNARELLA, ANDREA;BOLEGO, CHIARA
2013

Abstract

In response to various signals, macrophages undergo classical M1 activation (stimulated by toll-like receptor ligands and IFN-γ) or alternative M2 activation (stimulated by IL-4/IL-13). Functional polarization of macrophages has been observed in vivo under physiological and pathological conditions, and may represent an attractive target for pharmacological modulation. Given the lack of gold standards for M1/M2 activation in vitro, we polarized human monocyte-derived macrophages using different protocols and performed extensive characterization including the modulation of specific markers by dexamethasone. Human monocytes were obtained from healthy donor buffy coats by 2-step gradient centrifugation, and macrophages were obtained by culturing monocytes for 7 days in RPMI 1640 medium with 10% FCS. While virtually 100% of macrophages specifically stained for the intracellular glycoprotein CD68 on immunocytochemistry, only 10% showed surface expression as measured by flow cytometry, indicating a low basal activation state. When resting macrophages were incubated with LPS/IFN-γ (M1) for 4 h, no change was observed in the percentage of cells expressing the M1 surface markers CD68 and CCR2. After longer stimulation (48 h), the number of CD68- but not CCR2-positive cells doubled as compared with resting macrophages. This polarization protocol concomitantly down-regulated the expression of M2 markers CD206, CD163 and CX3CR1. In contrast, none of these markers were affected by alternative (M2) polarization with IL-4/IL-13 for 48 h whereas CD206 was up-regulated after 7 days. Q-PCR phenotyping after M1 polarization for 6-48 h showed increased TNF-α, IL-1β, COX-2, IL-10 and VEGF transcript levels with decreased CD206 and COX-1 expression. Finally, overnight pretreatment with 10 nM dexamethasone enhanced surface expression of CD163 in resting and M2-polarized macrophages, and reversed M1-induced CD163 down-regulation. We conclude that the phenotypic characterization of polarized macrophages is required and provides a basis for pharmacological macrophage targeting.
Inflammatory based diseases: molecular and cellular mechanisms
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2577333
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