M1 macrophages inhibit FRβ expression on M2-polarized macrophages and release high levels of activin A. (A) Cell-surface expression of FRβ on M1 and M2 macrophages determined by flow cytometry using a rabbit polyclonal antiserum against human FRβ (empty histogram) or a nonspecific rabbit preimmune antiserum (filled histogram). The percentage of marker-positive cells and the mean fluorescence intensity (in parentheses) are indicated. (B) FRβ function in M1 and M2 macrophages demonstrated by confocal microscopy on cells incubated at 37°C with folate-FITC (green fluorescence) and Transferrin-Texas red (red fluorescence). (C) FOLR2 mRNA expression levels determined by qRT-PCR in M2 macrophages after replacement of the culture supernatant by either M-CSF (gray histograms)- or GM-CSF (empty histograms)–containing complete medium for 48 hours. Results are expressed as relative mRNA levels (relative to GAPDH RNA levels and referred to the RNA levels in cells maintained in M-CSF–containing medium). The means and SD of triplicate determinations are shown; *P < .005. (D) FOLR2 mRNA expression levels determined by qRT-PCR on peripheral blood monocytes (Mo.) and monocytes cultured for 72 hours in the absence (0) or presence (M1 SN [%]) of increasing percentages of M1 macrophage–conditioned medium. Results are expressed as relative mRNA levels (relative to GAPDH RNA levels and referred to levels detected in Mo.). The means and SD of triplicate determinations are shown; *P < .005. (E) Relative INHBA gene expression in M1 and M2 macrophages determined by microarray DNA analysis (empty histograms) and qRT-PCR (gray histograms). (F) INHBA mRNA expression levels determined by qRT-PCR on the indicated macrophages after replacement of their respective culture supernatant with fresh complete medium containing either M-CSF (gray histograms) or GM-CSF (empty histograms) for 48 hours. The means and SD of triplicate determinations are shown. Results are expressed as relative mRNA levels (relative to GAPDH RNA levels) and referred to the INHBA RNA levels in cells treated with M-CSF; *P < .005. (G) Activin A levels released by M1 and M2 macrophages from 6 independent donors determined by ELISA. (H) Determination of activin A release during the differentiation of M1 and M2 macrophages from 2 independent donors determined by ELISA on culture supernatants removed at the indicated time points. (I) Activin A levels released by M1 and M2 macrophages untreated (−) or stimulated with 10 or 100 ng/mL LPS for 24 hours, and either maintained in their conditioned medium (no wash) or in fresh culture medium (wash) during the LPS stimulation period. (J) Activin A levels released by murine M1 and M2 BMDM from 4 independent samples determined by ELISA. For panels G-J, the means and SD of triplicate determinations are shown; P < .005; **P < .05.