ATP Evokes Ca2+ Responses and CXCL5 Secretion via P2X4 Receptor Activation in Human Monocyte-Derived Macrophages

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca 2+ ] i signals and cytokine production in response to ATP concentrations that exclude P2X 7 receptor activation. Expression of P2X 1 , P2X 4 , P2X 5 , P2X 7 , P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , and P2Y 13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca 2+ responses in a concentration-dependent fashion (EC 50 = 11.4 ± 2.9 μM, n = 3). P2Y 11 and P2Y 13 activations mediated the amplitude of [Ca 2+ ] i response, whereas P2X 4 activation, but not P2X 1 or P2X 7 , determined the duration of Ca 2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X 4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X 4 antagonists was lost in the absence of extracellular Ca 2+ Reciprocally, positive allosteric modulation of P2X 4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X 7 , P2Y 11 , or P2Y 13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X 4 in determining the duration of ATP-evoked Ca 2+ responses and CXCL5 secretion in human primary macrophage.