The scavenger activity of the human P2X7 receptor differs from P2X7 pore function by insensitivity to antagonists, genetic variation and sodium concentration: Relevance to inflammatory brain diseases

Abstract

Activation of P2X7 receptors is widely recognised to initiate proinflammatory responses. However P2X7 also has a dual function as a scavenger receptor which is active in the absence of ATP and plasma proteins and may be important in central nervous system (CNS) diseases. Here, we investigated both P2X7 pore formation and its phagocytic function in fresh human monocytes (as a model of microglia) by measuring ATP-induced ethidium dye uptake and fluorescent bead uptake respectively. This was studied in monocytes expressing various polymorphic variants as well as in the presence of different P2X7 antagonists and ionic media. P2X7-mediated phagocytosis was found to account for about half of Latrunculin (or Cytochalasin D)-sensitive bead engulfment by fresh human monocytes. Monocytes harbouring P2X7 Ala348Thr or Glu496Ala polymorphic variants showed increase or loss of ethidium uptake respectively, but these changes in pore formation did not always correspond to the changes in phagocytosis of YG beads. Unlike pore function, P2X7-mediated phagocytosis was not affected by three potent selective P2X7 antagonists and remained identical in Na and K media. Taken together, our results show that P2X7 is a scavenger receptor with important function in the CNS but its phagocytic function has features distinct from its pore function. Both P2X7 pore formation and P2X7-mediated phagocytosis should be considered in the design of new P2X7 antagonists for the treatment of CNS diseases.

P2X7 as a scavenger receptor for innate phagocytosis in the brain

“…These structural data have identified the inhibitory binding sites separate from previously identified ATP binding sites (Karasawa and Kawate, ; Allsopp et al ., ; Karasawa and Kawate, ). Thus, selective P2X7 antagonists may block the open pore state without affecting P2X7 scavenger activity, although oxidized ATP is an exception as it blocks both pore and scavenger function (Ou et al ., ). Although a dozen or more polymorphisms have been shown to affect the permeability of the open pore conformation, these polymorphisms have little or no effect on the scavenger activity of the P2X7 receptor.…”

“…However, none of the above three inhibitors reduced phagocytosis of YG beads by monocytes at inhibitor concentrations of 1 and 10 μM. In contrast, the earlier and less selective P2X7 inhibitor, KN62, reduced both YG bead uptake and ATP‐induced pore formation, perhaps as a results of its known inhibitory effects on Ca 2+ /calmodulin‐dependent kinase type II (CAMKII) (Ou et al ., ). These data show that the selective and potent P2X7 antagonists currently available are effective at blocking P2X7 pore formation but do not affect P2X7 phagocytosis in monocytes expressing wild‐type P2X7 receptors or the two common P2X7 functional variants.…”

“…Our recent study showed that three P2X7 antagonists, AZ10606120, A438079 and AF27139 completely abolished P2X7 pore formation at 1–10 μM without affecting the overall phagocytic function of monocytes, which were studied as surrogates for microglia (Ou et al ., ). Our data support the use of P2X7 antagonists for neurodegenerative diseases, in which the neuropathological features are accompanied by an inflammatory response (Heppner et al ., ).…”

“…Although the Thr 205 Met mutation is extremely rare in the population, these data suggest that a major reduction in the surface expression of P2X7 receptors may lead to impairment of P2X7-mediated innate phagocytosis in the brain. In contrast, common polymorphisms in P2X7 receptors, which cause either gain or loss of pore function have little effect on P2X7-mediated phagocytosis, with the exception of homozygosity of the Ala 348 Thr gain-of-function variant, which modestly increases monocyte phagocytic capacity (Ou et al, 2018). Further examination of the relationship between surface expression of P2X7 receptors and phagocytic function of the cell is needed.…”

Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNS

“…In apparent contradiction to this finding, neural progenitor cells and neuroblasts of human fetal telencephalon could clear apoptotic cells by innate phagocytosis mediated by their P2X7Rs [125]. It was astonishing that this scavenger activity was not prevented/reversed by pharmacological P2X7R antagonists [126].…”