ATP Evokes Ca2+ Responses and CXCL5 Secretion via P2X(4) 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 [Ca2+](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 Ca2+ responses in a concentration-dependent fashion (EC50 = 11.4 +/- 2.9 mu M, n = 3). P2Y(11) and P2Y(13) activations mediated the amplitude of [Ca2+](i) response, whereas P2X(4) activation, but not P2X(1) or P2X(7), determined the duration of Ca2+ 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 Ca2+. 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 Ca2+ responses and CXCL5 secretion in human primary macrophage.