Dynamic regulation of the P2X₄ receptor in alveolar macrophages by phagocytosis and classical activation

Abstract

ATP-gated P2X 4 receptors (P2X 4 R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X 4 R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X 4 R protein expression by Western blot and biotinylation assays and functional expression by whole-cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X 4 R expression by 2-to 7-fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN-c and TNF-a or IFN-c and LPS reduced surface and functional P2X 4 R expression by 3-fold without altering total P2X 4 R protein levels. Alternative activation with IL-4 or IL-13 did not alter total, surface or functional expression of P2X 4 R. This is the first study of the regulation of P2X 4 R in macrophages by physiological stimuli and presents a picture whereby P2X 4 R become functional in response to initial phagocytic stimuli but return to a non-functional state during sustained activation by classical macrophage activation.Key words: Inflammation . Ion channels . Patch-clamp . Purine receptors Introduction Extracellular ATP acts as a ''danger signal'' when it is released from cells during tissue damage and inflammation; it acts on metabotropic (G-protein coupled) P2Y and ionotropic (cation channel) P2X purinergic receptors to affect several inflammatory and immune responses [1,2]. There are seven members of the P2X receptor family (P2X 1-7 R) that show widespread distribution in both neuronal and non-neuronal tissues [3]. Two of these seven members, P2X 4 R and P2X 7 R, have generated increasing interest as potential anti-inflammatory drug targets [2,4,5]. The case for P2X 7 R is now well established: Stimulation of P2X 7 R in activated monocytes, macrophages and microglia leads to rapid release of pro-inflammatory IL-1b and IL-18 cytokines, to phospholipase D activation and, if stimulation is sustained, to apopotosis [1,[3][4][5]. Classical activation of macrophages, generally induced by IFN-g and/or LPS, up-regulates P2X 7 R mRNA and protein levels and functional expression [6,7] and this correlates well with a role for P2X 7 R in bacterial killing [8,9]. Recently, highly selective and potent P2X 7 R antagonists have been identified [10] with one of these showing initial positive results in Phase II clinical trials for rheumatoid arthritis [11]. The case for P2X 4 R remains tentative, largely due to the absence of selective P2X 4 R antagonists and the complicating presence of P2X 7 R, which is generally co-expressed with P2X 4 R in ...

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X7-KO mice

“…In our hands, to be effective, the activation of the plasma membrane receptors by the nucleotide had to be potentiated by ivermectin raising some doubts on their physiological role [37]. Stokes and Surprenant [24] have recently proposed a model to explain the contribution in vivo of the P2X 4 receptors to the physiology of macrophages. According to these authors, the activation of macrophages by a phagocytic stimulus promotes the translocation of P2X 4 receptors from the lysosomal compartment to the plasma membrane.…”

“…Brône and colleagues showed that ivermectin potentiated a current elicited by extracellular ATP in macrophages from WT mice but not from P2X 4 -KO mice [8]. Stokes and Surprenant [24] reported that ivermectin potentiated the current evoked by low concentrations of ATP in human alveolar macrophages. Our results are also fully consistent with recent results [25] who reported that ivermectin Fig.…”

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

“…Additionally, P2X4 receptors form functional heteromers with P2X6 subunits (Lê et al, 1998a;Ormond et al, 2006) and undergo interactions with P2X7 receptors (Guo et al, 2007;Nicke, 2008;Alqallaf et al, 2009;Boumechache et al, 2009;Casas-Pruneda et al, 2009;MurrellLagnado, 2009). Progress has also been made in understanding the cell biology of P2X4 receptors, with evidence to suggest that they undergo endocytosis and traffic to lysosomes Royle et al, 2002;Toulmé et al, 2006;Qureshi et al, 2007;Stokes and Surprenant, 2009). Early studies showed that P2X4 receptors were widely expressed in the brain (Lê et al, 1998b;Rubio and Soto, 2001), whereas more recent studies show that they are also expressed in microglia and alveolar macrophages (Bowler et al, 2003;Tsuda et al, 2003;Raouf et al, 2007;Ulmann et al, 2008;Stokes and Surprenant, 2009).…”

“…In addition to being present on the plasma membrane, some P2X receptors are also present within intracellular vesicular compartments (Ennion and Evans, 2001;Bobanovic et al, 2002;Toulmé et al, 2006;Fountain et al, 2007;Qureshi et al, 2007;Stokes and Surprenant, 2009). Seven P2X subunits comprise the P2X receptor family (P2X1-P2X7), with strong evidence to suggest that one type (P2X4) plays important roles in neuropathic pain (Tsuda et al, 2003;Coull et al, 2005).…”

The carbon monoxide donor, CORM-2, is an antagonist of ATP-gated, human P2X4 receptors

“…First cloned from rat brain [2,3], P2X4 subunits are expressed throughout the central nervous system, for example, in Purkinje cells [4], hippocampus [2] and dorsal horn of the spinal cord [5]. P2X4 is also widely expressed in non-neuronal tissues and can be found in the kidney, liver, pancreas [6], heart and lungs [7] oesteoclasts [8], and immune cells such as B lymphocytes [9] and macrophages [10]. In a great deal of these tissues and cells, the role of P2X4 receptors is not well understood.…”