The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.

IntroductionDuring the last few years the biologic activity of extracellular nucleotides has been the focus of increasing attention. Responses to extracellular adenosine triphosphate (ATP) or uridine triphosphate (UTP) as different as cell proliferation, differentiation, chemotaxis, cytokine secretion, release of lysosomal constituents, generation of reactive oxygen or nitrogen species, and cell death have been reported by different groups. [1][2][3][4][5][6][7][8][9] The effects of extracellular nucleotides are mediated by specific plasma membrane receptors, which are classified into 2 families: metabotropic P2Y receptors (P2YRs) 10 and ionotropic P2X receptors (P2XRs). [11][12][13] P2YRs are typical G protein-coupled receptors with 7 transmembrane domains. Eight P2YRs have been cloned so far. Signal transduction occurs via activation of phospholipase C or stimulation/ inhibition of adenylate cyclase. ATP is an agonist at all P2Y subtypes, with the exception of P2Y 12 and P2Y 14 , where adenosine diphosphate (ADP) and uridine diphosphate (UDP)-glucose are the preferred agonists, respectively, and ATP is either ineffective or an antagonist. UTP is a better agonist than ATP at P2Y 4 and P2Y 6 , whereas at P2Y 2 ATP and UTP are equipotent. P2XRs are multimeric plasma membrane ion channels directly gated by ATP that mediate fast permeability changes to monovalent and divalent cations (Na ϩ , K ϩ , and Ca 2ϩ ). Seven P2X subunits have been cloned so far. P2Rs are widely expressed on mature blood cells. [14][15][16][17][18][19][20] Moreover, the expression and function of the P2X 7 R on B-cell chronic lymphocytic leukemia (B-CLL) cells correlate with the severity of the disease, and targeting of such receptors has been proposed as a novel form of treatment for chronic leukemia. 21 No information is currently available on the expression and function of P2Rs on human hematopoietic stem cells (HSCs). HSCs are identified by the expression of the CD34 antigen, a cell membrane phosphoglycoprotein present on human bone marrow (BM), peripheral blood (PB), and cord blood progenitors. 22 Animal models 23 and clinical transplantation studies 24 using highly enriched cell populations have demonstrated the capacity of CD34 ϩ cells to behave as true stem cells because they engraft lethally irradiated allogeneic hosts. Moreover, retrovirally marked autologous CD34 ϩ grafts sustain long-term hematopoiesis. 25 Recent studies have challenged the dogma that all HSCs express the CD34 antigen. [26][27][28][29][30] Sato and colleagues 31 have demonstrated that only one class of murine stem cell exists, although in 2 functional states distinguishable by CD34 expression. According to this model, CD34 Ϫ stem cells are quiescent and can be activated by different stimuli to generate a CD34 ϩ cell population with high engraftment potential. 32,33 In this study, we assessed the expression of P2Rs on highly purified CD34 ϩ HSCs and investigated the functional responses of Materials and methods ReagentsATP, UTP, cytidine triphosphate (CTP), guano...

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X7R in HEK293 and K562 cells transfected with P2X7 cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X7-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X7 stimulation. In particular, ATP-mediated Ca2+ influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X7 were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X7R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.

ATP is abundantly released from stressed or damaged cells in response to mechanical stimulation, bacteria, or noxious agents. In this study, we have investigated the possible involvement of P2 receptors (receptor for extracellular nucleotides) in the expression and release of inflammatory mediators by human keratinocytes. Notably, extracellular ATP displayed a complex regulation of IFN-gamma-stimulated chemokine expression, with upregulation of CC chemokine ligand 2 (CCL2), CCL5 and CXC chemokine ligand 8 (CXCL8), and suppression of the receptor CXC chemokine receptor 3 (CXCR3), CXCL9, CXCL10, and CXCL11. The effect of ATP was mimicked by ADP and adenosine-5'-O-3-thiotriphosphate, whereas 2',3'-O-(4-benzoylbenzoyl) ATP (BzATP) downmodulated all chemokines investigated. UTP had no effect on IFN-gamma-stimulated chemokine secretion. The broad-spectrum P2 receptor antagonist suramin and the selective P2Y1 inhibitor adenosine 3'-phosphate 5'-phosphosulfate counteracted the effect of ATP on secretion of all the chemokines examined, whereas pyridoxal phosphate 6-azophenyl 2',4'-disulfonic acid and KN62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl] 4 phenylpiperazine) partially prevented the inhibitory effect of ATP on CXCL10 secretion, but on the other hand potentiated the ATP-stimulatory effect on CCL5, CCL2, and CXCL8 release. In lesional skin of psoriasis and atopic dermatitis patients, intense P2X7 reactivity was confined to the cell membrane of the basal layer, whereas diffuse P2Y1 immunostaining was found throughout the epidermis. Collectively, our data suggest that the orchestrated activation of distinct P2Y and P2X receptors modulates skin inflammation.

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. A loss-of-function single nucleotide polymorphism (SNP) at position 1513 (1513 A-->C) of the P2X(7) gene has recently been identified in both healthy and chronic lymphocytic leukemia (CLL) B-cells, translating into a loss of P2X(7)-mediated apoptosis in these cells. This antiapoptotic effect results in increased B-cell numbers, thereby potentially contributing to the survival of B-CLL clones. It was hypothesized that prolonged cell survival may also predispose to induction of autoimmunity. The objective of this study is to analyze the role of the P2X(7) receptor and its loss-of-function 1513 A-->C polymorphism (SNP) in the development of systemic lupus erythematosus (SLE). DNA samples obtained from patients with sporadic SLE were analyzed for the presence of the 1513 A-->C polymorphism using polymerase chain reaction (PCR) amplification and then direct sequencing. No significant difference in allele frequencies (1513 A-->C polymorphism) between sporadic cases of SLE and controls was found. A loss-of-function SNP at position 1513 (1513 A-->C) of the P2X(7) gene does not appear to be a susceptibility gene locus for the development of sporadic SLE.