Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

1) Does the effect of extracellular adenosine fully account for the anti-inflammatory effect of apoptotic cells on macrophages that take them up, or only to a fraction of it? This can be answered by determining the actual concentrations of AMP and adenosine in the co-culture of apoptotic cells and macrophages and then comparing the extent of anti-inflammatory effect of the apoptotic cells (as assessed for example by quantifying the upregulation of one of the anti-inflammatory genes in the macrophages) to the anti-inflammatory effect of AMP and adenosine, when applied to the macrophages at those same concentrations, yet in the absence of apoptotic cells.

Cells expressing the caspase-resistant form of pannexin-1 do not release AMP during FasL-treated apoptosis (Figure 4B). To address the above question, we treated the parental W3 and the W3 cell transformants expressing the caspase-resistant pannexin1, and co-cultured for 1 h with Bone-marrow derived macrophages (BMDM). The Thbs1 gene expression in BMDM was then quantified by real-time PCR, and was found that BMDM co-cultured with the FasL-treated wild-type W3 cells strongly express Thbs1. But, this was not observed when BMDM was co-cultured with FasL-treated W3 cells expressing the caspase-resistant form of Pannexin 1. The result was shown in Figure 4C of the revised manuscript and explained.

2) Is the extracellular adenosine in co-cultures of apoptotic cells and macrophages indeed fully derived from the apoptotic cells or, as was suggested previously by Köröskényi et al., in part also derived from the mononuclear phagocytes that take them up. An answer to the latter question can be obtained by comparing the concentrations of AMP+ Adenosine in cultures at which the mononuclear phagocytes are allowed to take up apoptotic cells to those in cultures of the same cells in which such uptake is prevented, for example by a semipermeable barrier.

To address the above question whether the macrophages engulfing apoptotic cells produce AMP or not, we first incubated mouse resident peritoneal macrophages (5 x 10⁵ cells) with FasL-treated mouse thymocytes (2 x 10⁶ cells) for 1 h for engulfment. The macrophages were thoroughly washed with medium to remove apoptotic cells, and cultured for 5 h. The AMP level increased in the supernatant was about 30 nM, corresponding to 30 pmoles per 5 x 10⁵ cells. This value is significantly lower than that produced by apoptotic cells (7 nmols per 10⁷ cells) (Figure 4A). This point was briefly mentioned in the Discussion.