To analyze whether pro-inflammatory signaling is affected in Ptdsr -/- macrophages, we stimulated FLDMs from Ptdsr+/+ and Ptdsr -/- mice with LPS and measured levels of tumor necrosis factor-α (TNF-α) at different time points after stimulation (Figure 8c). Ptdsr -/- macrophages produced significantly less TNF-α than did wild-type macrophages. The difference in TNF-α secretion was first visible after 3 h of LPS stimulation and became more prominent during the course of the experiment (for example, after 9 h and 12 h of LPS stimulation; Figure 8c). To analyze whether TNF-α release by Ptdsr -/- macrophages can be affected by engulfment of apoptotic cells, we stimulated FLDMs with LPS, apoptotic cells or both. Quantification of TNF-α levels by ELISA after 22 h showed that Ptdsr-deficient macrophages release less TNF-α after stimulation with LPS alone, and also after double stimulation of macrophages with LPS and apoptotic cells (Figure 8d). Moreover, the double stimulation demonstrated that the LPS-induced TNF-α release by Ptdsr -/- macrophages could be inhibited by co-administration of apoptotic cells to an extent comparable to that seen in wild-type macrophages. Similar results were obtained when other pro-inflammatory cytokines, such as interleukin-6 and monocyte chemoattractant protein-1, were analyzed (data not shown). These results indicate that Ptdsr is not required in macrophages for the inhibition of pro-inflammatory signaling after recognition and engulfment of apoptotic cells. Ptdsr-deficiency does, however, affect the overall release of pro- and anti-inflammatory cytokines after stimulation with LPS and after double treatment with LPS and apoptotic cells, indicating that Ptdsr-deficient macrophages have a reduced capacity to produce or secrete pro- and anti-inflammatory cytokines.