Figure 5.
Hypoxia forces M2 differentiation of CLL monocytes through the A2A adenosine receptor. (A) Representative Giemsa staining (left panels) obtained after 12- to 14-day cultures of PBMCs from 8 CLL patients. Where indicated, cultures were performed in the presence of the A2A agonist CGS21680 or antagonist SCH58261 (both 1 µM, added every 48 hours). Macrophage differentiation was confirmed by staining with the lineage-specific marker CD68 (green). Slides were counterstained with Alexa Fluor 568–conjugated phalloidin (red) and DAPI (blue). Expression of the M2 macrophage markers CD206 (green) and CD163 (red) was evaluated in the same samples. Original magnification ×10 (Giemsa staining) and ×63 (confocal microscopy). Scale bars represent 50 and 25 µm for the CD68 and CD206/CD163 panels, respectively. (B) Boxplots show cumulative mean pixel intensity data in CD206 (left) and CD163 (right) obtained from at least 5 measurements in 3 different random fields for each of the 6 samples analyzed. (C-F) RT-PCR data showing expression of the interferon regulatory factor 4 (IRF4; C) transcription factor, the enzyme indoleamine 2,3-dioxygenase (IDO; D), the CCL3 chemokine (CLL3; E), and IL-6 (IL6; F) in NLC preparations obtained in 21% or 1% O2, in the presence of A2A agonists and antagonists (both at 1 µM, added every 48 hours throughout the 12- to 14-day differentiation process, n = 10). (E) Boxplot on the right represents cumulative data of the migration index (MI) calculated on CD14+ cells from normal donors in the presence of NLC spent media. (F) Increased expression of IL-6 was confirmed by checking protein concentrations in culture supernatants using an ELISA assay (right panel). (G) Boxplot showing the percentage of live CLL cells in coculture with NLCs obtained under normoxic or hypoxic conditions. After the differentiation period, autologous purified CLL cells were thawed and plated over the NLC layer and cultured for 48 hours under normoxia or hypoxia. Apoptosis was evaluated by staining with Annexin V and propidium iodide (n = 7). The Wilcoxon signed rank and Mann-Whitney U tests, followed by the Tukey test, were used for statistical analyses. CGS, CGS21680; H, hypoxia (gray boxes); N, normoxia (open boxes); SCH, SCH58261.

Hypoxia forces M2 differentiation of CLL monocytes through the A2A adenosine receptor. (A) Representative Giemsa staining (left panels) obtained after 12- to 14-day cultures of PBMCs from 8 CLL patients. Where indicated, cultures were performed in the presence of the A2A agonist CGS21680 or antagonist SCH58261 (both 1 µM, added every 48 hours). Macrophage differentiation was confirmed by staining with the lineage-specific marker CD68 (green). Slides were counterstained with Alexa Fluor 568–conjugated phalloidin (red) and DAPI (blue). Expression of the M2 macrophage markers CD206 (green) and CD163 (red) was evaluated in the same samples. Original magnification ×10 (Giemsa staining) and ×63 (confocal microscopy). Scale bars represent 50 and 25 µm for the CD68 and CD206/CD163 panels, respectively. (B) Boxplots show cumulative mean pixel intensity data in CD206 (left) and CD163 (right) obtained from at least 5 measurements in 3 different random fields for each of the 6 samples analyzed. (C-F) RT-PCR data showing expression of the interferon regulatory factor 4 (IRF4; C) transcription factor, the enzyme indoleamine 2,3-dioxygenase (IDO; D), the CCL3 chemokine (CLL3; E), and IL-6 (IL6; F) in NLC preparations obtained in 21% or 1% O2, in the presence of A2A agonists and antagonists (both at 1 µM, added every 48 hours throughout the 12- to 14-day differentiation process, n = 10). (E) Boxplot on the right represents cumulative data of the migration index (MI) calculated on CD14+ cells from normal donors in the presence of NLC spent media. (F) Increased expression of IL-6 was confirmed by checking protein concentrations in culture supernatants using an ELISA assay (right panel). (G) Boxplot showing the percentage of live CLL cells in coculture with NLCs obtained under normoxic or hypoxic conditions. After the differentiation period, autologous purified CLL cells were thawed and plated over the NLC layer and cultured for 48 hours under normoxia or hypoxia. Apoptosis was evaluated by staining with Annexin V and propidium iodide (n = 7). The Wilcoxon signed rank and Mann-Whitney U tests, followed by the Tukey test, were used for statistical analyses. CGS, CGS21680; H, hypoxia (gray boxes); N, normoxia (open boxes); SCH, SCH58261.

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