F Di Virgilio

The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation.

To cite this article: Müller T, Robaye B, Vieira RP, Ferrari D, Grimm M, Jakob T, Martin SF, Di Virgilio F, Boeynaems J‐M, Virchow JC, Idzko M. The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation. Allergy 2010; 65: 1545–1553.

Extracellular purines, purinergic receptors and tumor growth.

Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host–tumor interaction and highlights novel therapeutic options stemming from recent advances in this field.

High glucose modulates P2X7 receptor-mediated function in human primary fibroblasts

Aims/hypothesis. Purinergic receptors are a family of newly characterized plasma membrane molecules involved in several and as yet only partially known cellular functions such as vascular reactivity, apoptosis and cytokine secretion. Little is known about the effect extracellular microenvironment has on their function. Fibroblasts share several features with smooth muscle cells and are an important constituent of the atherosclerotic plaque. Our aim was to evaluate the effect of high glucose concentration on ATP-mediated responses in human fibroblasts.¶Methods. Fibroblasts were obtained by skin biopsies and grown at two different glucose concentrations. We evaluated receptor expression by RT-PCR and immunoblotting and receptor localization by immunofluorescence. Plasma membrane potential and calcium changes were measured by fluorescent indicators. Apoptosis was determined by ethidium bromide staining and caspase-3 activation.¶Results. We show that cells grown in a medium with high glucose concentration underwent great ATP-mediated morphological changes, enhanced apoptosis, caspase 3 activation and interleukin-6 release. We identified P2X7 as the main purinergic receptor involved in these responses. Furthermore, high glucose concentration triggered the assembly of P2X7 into ring-like structures located at the periphery of the cells.¶Conclusion/interpretation. Given that ATP is frequently released into the extracellular milieu upon cell and tissue damage, secretory exocytosis or activation of plasma membrane transporters, we hypothesize that ATP receptors participate in the pathogenesis of vascular complications of diabetes. [Diabetologia (2000) 43: 1248–1256]

The P2X7 receptor is a key modulator of aerobic glycolysis.

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment.

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b+/Gr-1+ cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1+ population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule

P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro‐inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown.

On the Role of the P2X7 Receptor in the Immune System

Twenty years ago Gomperts and Cockcroft were the first to describe ATP-dependent permeability increases in a cell type involved in the immune response (mast cells), hypothesizing that “a possible mechanism would involve channel formation by the aggregation of transmembrane monomeric units…” (Cockcroft and Gomperts 1979). Ever since, similar responses to ATP have been described in many other immune and inflammatory cells (356-1|Table 1), leading many authors to propose that expression of the “ATP permeabilizing receptor” is a feature of cells involved in host defense. In mast cells, as well as in other immune cells, the active form was found to be ATP in its fully dissociated form. Accordingly, this hypothetical receptor was named the ATP4-receptor, later to become P2Z when Gordon (1986) carefully defined the properties of the P2 receptor of mast cells and lymphocytes, and found that this receptor did not entirely fit the P2X/P2Y subclassification originally proposed by Burnstock and Kennedy (1985). The functional “signature” of the P2Z receptor (reversible permeabilization of the plasma membrane) was so peculiar that many doubted the actual “receptor” nature of P2Z, reckoning that ATP might cause in immune cell types a nonspecific perturbation of the plasma membrane that in turn led to permeability transition. However, even before the cloning of the actual molecule responsible for ATP-dependent permeabilization, evidence supporting the receptor nature of P2z was compelling as: 1. Other nucleosides or nucleotides could not mimic this effect, even at high concentrations (Cockcroftand Gomperts 1980; Steinberg et al. 1987). 2. It was possible to select from ATP-sensitive lines cell clones fully resistant to ATP (Steinberg and Silverstein 1987; Murgia et al. 1992). 3. Periodate-oxidized ATP (oATP) was shown to completely block permeabilization, and another ATP analog, 2-methylthio-9-β-l-ribofuranosyladenine 5′-triphosphate (2-MeS-L-ATP) was 50% inhibitory (Murgia et al. 1993; Tatham et al. 1988). Table 1 Cells expressing the P2X7/P2Z receptor Cell type Method References Rat mast cells Functional responses Cockcroft and Gomperts (1979) Mouse T and B lymphocytes Functional responses (?) Di Virgilio et al. (1989); Filippini et al. (1990a,b); Chused et al. (1996) Human B lymphocytes Functional responses (?), Abs, RT-PCR Wiley and Dubyak (1989); Ferrari et al. (1994); Bretschneider et al.(1995); O.R. Baricordi et al. (in preparation) Human T lymphocytes Functional responses (?); RT-PCR Baricordi et al. (1996); O.R. Baricordi et al. (in preparation) Mouse macrophages Functional responses, cloning, Abs, in-situ hybridization, RT-PCR Steinberg et al. (1987);Surprenant et al. (1996); Chiozzi et al. (1997) Human macrophages Functional responses.cloning, Abs, RT-PCR Hickman et al. (1994);Falzoni et al. (1995);Dubyak et al. (1996); Rassendren et al. (1997) Mouse/rat microglial cells Functional responses.Abs, in situ hybridization Ferrari et al. (1996,1997a);Collo et al. (1997) Human Langerhans cells Functional responses Girolomoni et al. (1993) Human dendritic cells Abs Buell et al. (1998) Mouse dendritic cells Functional responses, Abs Mutini et al. (1999) Human fibroblasts Functional responses, Abs, RT-PCR Solini et al. (1999) Porcine and bovine endothelial cells Functional responses.RT-PCR Von Albertini et al. (1998) Rat retina neurons Functional responses, Abs, RT-PCR Brandle et al. (1998) Rat mesangial cells Functional responses, Northern Schulze-Lohoff et al. (1998) CHO cells Functional responses.RT-PCR Michel et al. (1998) Rat supraoptic neurons RT-PCR Shibuya et al. (1999) Rat salivary gland RT-PCR Turner et al. (1998) Rat submandibular glands Functional responses RT-PCR Alzola et al. (1998) Rat parotid acinar cells Functional responses.RT-PCR Tenneti et al. (1998) Rat hepatic arteries RT-PCR Phillips et al. (1998) Human saphenous vein smooth muscle Functional responses, RT-PCR Cario-Toumaniantz et al. (1998)

Nimodipine inhibits IL‐1β release stimulated by amyloid β from microglia

BACKGROUND AND PURPOSE There is growing evidence that inflammation plays a major role in the pathogenesis of neural damage caused by deposition of amyloid β (Aβ) in the brain. Nimodipine has received attention as a drug that might improve learning and reduce cognitive deficits in Alzheimers disease, but the mechanism of action is poorly known. In this study, we tested the hypothesis that nimodipine inhibited Aβ‐stimulated IL‐1β release from microglia.

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phosphatidylinositol turnover measured as modification of incorporation of [32P]Pi and [3H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca2+, without a decrease in the total quantity of Ca2+ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca2+ concentration [Ca2+]i, without a decrease in the magnitude of the final increase of [Ca2+]i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca2+-phospholipid-dependent protein kinase C.

P2X7 gene polymorphisms do not appear to be a susceptibility gene locus in sporadic cases of systemic lupus erythematosus

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.