P2X4 receptor regulates P2X7 receptor-dependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells

Activation of P2X7 receptor of dendritic cells plays a significant role in inflammation through production of cytokines such as IL-1β, and recent studies have suggested structural and functional interactions of P2X7 receptor with P2X4 receptor in macrophages. However, it is unknown whether P2X4 receptor modulates P2X7 functions in dendritic cells. Here, we present evidence that expression of P2X4 receptor is required for P2X7 receptor-dependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells (BMDCs). We confirmed expression of both P2X7 receptor and P2X4 receptor in BMDCs. Treatment of BMDCs with 3 mM ATP caused a transient, P2X4-dependent elevation, or spike, of intracellular Ca(2+) level [Ca(2+)]i, followed by the sustained P2X7-dependent increase of [Ca(2+)]i. We performed knockdown of P2X4 receptor in BMDCs by transfection with short hairpin RNA targeting this receptor. The ATP-induced initial peak of [Ca(2+)]i was decreased in P2X4-knockdown cells (P2X4-KD). Further, we found that ATP-induced IL-1β and IL-18 release from LPS-primed BMDCs was suppressed by pretreatment with P2X7 antagonist A438079 or P2X4 antagonist TNP-ATP. The P2X7-dependent IL-1β and IL-18 release was significantly lower in P2X4-KD cells. Chelation of intracellular Ca(2+) also caused suppression of ATP-induced IL-1β and IL-18 release. These results suggest that P2X4 receptor-induced Ca(2+) influx is required for effective production of IL-1β and IL-18 via activation of P2X7 receptor in BMDCs. We conclude that co-expression of P2X4 receptor with P2X7 receptor in dendritic cells leads to enhancement of inflammation through facilitation of P2X7-dependent release of pro-inflammatory cytokines.