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 [Ca²⁺]_i signals and cytokine production in response to ATP concentrations that exclude P2X₇ receptor activation. Expression of P2X₁, P2X₄, P2X₅, P2X₇, P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, and P2Y₁₃ was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca²⁺ responses in a concentration-dependent fashion (EC₅₀ = 11.4 ± 2.9 microM, n = 3). P2Y₁₁ and P2Y₁₃ activations mediated the amplitude of [Ca²⁺]_i response, whereas P2X₄ activation, but not P2X₁ or P2X₇, determined the duration of Ca²⁺ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X₄ 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 P2X₄ antagonists was lost in the absence of extracellular Ca²⁺ Reciprocally, positive allosteric modulation of P2X₄ (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X₇, P2Y₁₁, or P2Y₁₃ receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X₄ in determining the duration of ATP-evoked Ca²⁺ responses and CXCL5 secretion in human primary macrophage.