Induction of the P2z/P2X₇ Nucleotide Receptor and Associated Phospholipase D Activity by Lipopolysaccharide and IFN-γ in the Human THP-1 Monocytic Cell Line

The activation of phospholipase D (PLD) was used as a marker for P2z nucleotide receptor (P2zR) activity in the human THP-1 monocytic cell line. While untreated THP-1 cells displayed little PLD response to the P2zR agonist 3′-O-(4-benzoyl)benzoyl (Bz)ATP, treatment with either IFN-γ or bacterial LPS induced a BzATP-mediated stimulation of PLD activity. Treatment of cells with combinations of IFN-γ and LPS resulted in synergistic induction. P2z receptors mediated these effects because: 1) reduction of extracellular divalent cation concentration increased agonist potency; 2) only BzATP or ATP acted as agonist nucleotides; 3) oxidized ATP, an inhibitor of the P2z receptor, abolished the response. The P2zR-stimulated PLD was rapidly activated (t_1/2 = 1.5 min), completely inhibited by KN-62, a calcium-calmodulin kinase II inhibitor, and only partially repressed by bisindolylmaleimide, a protein kinase C inhibitor. The P2zR-mediated PLD activity was distinguished from phorbol ester-stimulated PLD activity because the latter was slowly activated (t_1/2 > 15 min), unaffected by oxidized ATP or KN-62, and completely inhibited by bisindolylmaleimide. IFN-γ and LPS treatment also synergistically induced P2zR-dependent changes in membrane permeability and cytolysis, as indicated by BzATP-mediated Ca²⁺ influx, ethidium bromide uptake, and lactate dehydrogenase release. Finally, IFN-γ and LPS synergistically up-regulated mRNA encoding the P2X₇ receptor, a recently cloned ATP-gated channel that exhibits a P2zR phenotype.