P2Y6 receptor inhibition perturbs CCL2-evoked signalling in human monocytic and peripheral blood mononuclear cells

The chemokine CCL2 serves to target circulating monocytes and other leukocytes to tissue during innate immune responses and the progression of chronic inflammatory disease via activation of CCR2 receptor. Here we show that co-activation of the P2Y6 purinergic receptor occurs when THP-1 cells and human peripheral blood mononuclear cells sense CCL2 through CCR2. Furthermore, P2Y6 receptor activation accounts for approximately 80% of the intracellular calcium signal evoked by CCL2. Scavenging extracellular nucleotides with apyrase caused a 4-fold reduction in THP-1 sensitivity to CCL2 whereas inhibition of CD39-like ectonucleotidases potentiated CCL2-evoked calcium responses. Pharmacological inhibition of P2Y6 impairs CCL2-evoked calcium signalling and chemotaxis in peripheral blood mononuclear cells and THP-1 cells. Furthermore, stable P2Y6 knockdown (2-fold) in THP-1 cells impairs CCL2-evoked calcium signalling, chemotaxis and adhesion to TNFα-treated HUVECs. We demonstrate that THP-1 cells rapidly secrete ATP during signalling on the CCL2-CCR2 axis and suggest this may act as a mechanism for P2Y6 co-activation following CCL2 activation of the CCR2 receptor. The discovery that P2Y6 mediates leukocyte responsiveness to CCL2 represents a novel mechanism with which to modulate CCL2 signals.