Fig. 5
In vitro induced TAM-like macrophages show M2-polarization, arginase activity and ectonucleotidase expression. Peripheral blood monocytes were obtained from healthy volunteers by gradient centrifugation and subsequent adherence enrichment. Monocytes were then matured in Lumox dishes for 9 days to obtain mature macrophages (MФ). Macrophage polarization was achieved either with IFN-γ [1 μg/ml] and LPS [10 μg/ml] (MФ (IFN-γ + LPS)) or by coincubation with OAW-42 OvCA cells (MФ (Co-culture)). a The obtained MФ (IFN-γ + LPS) (light grey) and MФ (Co-culture) (dark grey) were analyzed by FACS for intracellular IL-10 and IL-12 levels. To obtain specific fluorescence intensity (SFI) values, the median fluorescence values obtained with fluorochrome-conjugated specific antibodies were divided by the median fluorescence values obtained with identically labeled irrelevant isotype-matched control antibodies (n = 3, * for p < 0.05, ** for p < 0.01). b CD14+ cells (TAM) were isolated from malignant tumor tissue. In addition, mature macrophages from a healthy donor were polarized as in a. To determine arginase activity by MФ (IFN-γ + LPS) and MФ (Co-culture) as well as by TAM, arginine conversion was assessed by measuring the resulting urea in a colorimetric assay. Significance levels were determined by two-sided, unpaired Student’s t-test. p-values < 0.01 were considered as highly significant (**). c Macrophages were prepared and polarized as in a. 8 h, 24 h and 48 h after the polarizing conditions had been applied, RNA was isolated, reverse-transcribed to cDNA and analyzed for CD39 and CD73 transcript levels using SybrGreen-based RT-PCR. 18S rRNA content was determined for normalization and relative CD39 and CD73 mRNA levels were calculated by the ΔΔCt method using non-polarized macrophages as reference. d Using MФ (IFN-γ + LPS) and MФ (Co-culture) as in c, CD39 and CD73 surface expression was assessed by flow cytometry. e Macrophages were polarized with OAW-42 cells for 24 h in the presence or absence of the A2A adenosine receptor inhibitor SCH58261 before IL-10, CD39 and CD73 expression were analysed by flow cytometry. Comparable data for primary TAMs isolated from OvCA ascites are shown in Fig. 2