New Role for ATP P2X¹ Receptors in the Control of Neutrophil Apoptosis and Respiratory Burst Activity.

P2X receptors are membrane non-selective cation channels that open in response to the binding of extracellular ATP. Seven genes encode P2X receptor subunits (P2X_1–7) in vertebrates. Channels form homo- or hetero-multimers of several subunits that are highly permeable to Ca²⁺. P2X receptors are widely distributed. In recent years, increasing attention has been paid to extracellular ATP as a candidate danger signal locally released at the inception of inflammation. One of the most striking features of ATP is its ability to promote P2X₇ receptor-mediated massive release of mature IL-1β from LPS-primed mononuclear phagocytes. On neutrophils, ATP rises intracellular Ca²⁺ concentrations, contributes to degranulation, adhesion, oxidative burst and delays apoptosis, events that may partly depend on the G-protein coupled P2Y₂ receptor and on P2X₇. In the present work, RT-PCR, Western blotting and immunofluorescence experiments reveal that neutrophils also express P2X₁ and P2X₅ receptor subtypes. A microarray analysis indicates that a 3-hour treatment of human peripheral blood neutrophils with the selective P2X₁ and P2X_1/5 receptor agonist, a,β-meATP, changes the expression of genes mainly involved in the control of cell fate. Accordingly, this agonist causes an increase of phosphatidylserine exposure on neutrophil membranes, maximally occurring after 3 hours and lasting until 18 hours of culture. In the presence of the protein synthesis inhibitor cycloheximide, a,β -meATP promotes caspase-3-dependent neutrophil apoptosis after 3 hours, which is correlated with highly reduced Fcγ RIII (CD16) membrane expression. In addition, a 1 min pretreatment of neutrophils with a,β -meATP potently increases tumor necrosis factor-a (TNF-α )-driven priming (30 min) of the respiratory burst induced by the bacterially derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Furthermore, a,β -meATP produces L-selectin (CD62L) shedding by its own and in an additive manner with TNF-α . This agonist also induces rapid and reversible phosphorylation of the survival kinases ERK (starting after 2 min) and Akt (15 min) as well as phosphorylation and degradation (after 2 min) of I-κ Bα , an inhibitor of the anti-apoptotic transcription factor NF-κ B. Hence, neutrophil P2X₁ receptors might have a dual role in inflammation; they would both contribute to neutrophil activation and promote cell death of neutrophils that have reached the end of their useful life span. Activation of P2X₁ receptors by extracellular ATP may thus represent novel regulatory mechanisms that govern neutrophil function.