Expression of adenosine receptors on human monocytes, immature, mature, and adenosine-differentiated DCs and the effect of adenosine on DC differentiation. (A) CD14⁺ monocytes were purified from peripheral blood; immature DCs (iDCs) were generated from monocytes cultured in the presence of GM-CSF and IL-4 for 5 days without (Control iDC) or with NECA (Adenosine iDC); for maturation 1 μg/mL LPS was added to immature DCs for 2 additional days (DC). Expression of adenosine receptor mRNA transcripts was assessed by real-time PCR as described in “Methods.” Average values from 4 different experiments are shown. Error bars denote SE. (B) Addition of adenosine or its stable analog NECA skews DC differentiation from monocytes toward a CD1a^lowCD14⁺ cell population. Monocytes were differentiated into immature DCs as above in the absence (Control iDC) or in the presence of 100 μM adenosine plus 10 μM EHNA (Adenosine iDC) or 100 μM NECA (NECA iDC). Combination of adenosine and the adenosine deaminase inhibitor EHNA was used to decelerate adenosine catabolism in cell culture. Expression of CD1a⁺ and CD14⁺ markers was assessed by flow cytometry; results are representative of 7 experiments. (C) Concentration-dependence curves of the generation of CD1a⁺ and CD1a^lowCD14⁺ cells in the presence of NECA. Concentrations of NECA corresponding to 50% of maximal effects are shown. Average values from 3 different experiments are shown. Error bars denote SE.