Juana M. Sanz

Kinetics and Mechanism of ATP-Dependent IL-1β Release from Microglial Cells

Endotoxin-dependent release of IL-1β from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1β is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1β. ATP had no effect on the accumulation of intracellular pro-IL-1β in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1β. Optimal ATP concentration for IL-1β secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1β release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1β release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-β-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-β by activating the IL-1-β-converting enzyme/caspase 1.

Cytolytic P2X purinoceptors

Anedoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloride-sensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.

Activation of Microglia by Amyloid β Requires P2X7 Receptor Expression

Extracellular ATP is a mediator of intercellular communication and a danger signal. Release of this and other nucleotides modulates microglia responses via P2Y and P2X receptors, among which the P2X7 subtype stands out for its proinflammatory activity and for up-regulation in a transgenic model of Alzheimer disease and in brains from Alzheimer disease patients. Here we show that amyloid β (Aβ) triggered increases in intracellular Ca2+ ([Ca2+]i), ATP release, IL-1β secretion, and plasma membrane permeabilization in microglia from wild-type but not from P2X7-deleted mice. Likewise, intra-hippocampal injection of Aβ caused a large accumulation of IL-1β in wild-type but not in P2X7−/− mice. These observations suggest that Aβ activates a purinergic autocrine/paracrine stimulatory loop of which the P2X7 receptor is an obligate component. Identification of the P2X7 receptor as a non-dispensable factor of Aβ-mediated microglia stimulation may open new avenues for the treatment of Alzheimer disease.

Chapter 29 The P2Z/P2X7 receptor of microglial cells: A novel immunomodulatory receptor

Publisher Summary This chapter investigates the role of the P2X7 receptor, formerly also known as P2Z, in microglia activation. The chapter presents the first evidence in support of the expression by microglial cells of an ATP-gated plasma membrane pore closely resembling the macrophage P2Z receptor. This was inferred on the basis of the ability of ATP to cause (1) fast plasma membrane depolarization, (2) uptake of ethidium bromide or lucifer yellow, and (3) release of cytoplasmic markers (e.g., lactate dehydrogenase), responses generally accepted as functional evidence for the expression of P2Z. This strong but indirect evidence was then confirmed by immunoblot analysis with an antiserum raised against the COOH tail of the P2X7 receptor that is in the meantime cloned from a rat brain library. A detailed investigation in the CNS gave no evidence for the presence of P2X7 in central neurons, thus suggesting that this receptor might have been originally cloned from microglia. The finding of a P2Z/P2X7 type receptor in microglia is consistent with the known peculiar expression of this molecule by immune cells and by mononuclear phagocytes.

Purinergic P2X7 receptor: A pivotal role in inflammation and immunomodulation

Many, potentially all, inflammatory and immune cells express purinergic receptors of the P1 and P2 type. In recent years, availability of molecular probes and, in some cases, specific antibodies has initiated an investigation of their possible involvement in the inflammatory response. A very recent acquisition is that, besides expressing purinergic receptors, immune cells also secrete ATP, that, once in the extracellular milieu, undergoes hydrolysis by various hydrolases or kinases. Besides active secretion, ATP can also leak into the pericellular space as a consequence of cell damage. Although adenosine appears to have a preeminent anti‐inflammatory activity, ATP by means of the P2X7 receptor seems to act as a proinflammatory mediator, a cytotoxic factor, or both. The combined activity of adenosine and ATP provides means for a fine modulation of the inflammatory response, depending on the amount of extracellular ATP, the rate of hydrolysis and the level of expression of P2 and P1 receptors. The P2X7 receptor appears to be involved in several processes relevant to inflammation (interleukin‐1β release, cytotoxicity, formation of macrophage polykarions), thus, it may be an appealing target for pharmacologic intervention. Drug Dev. Res. 45:207–213, 1998.

The P2X7 receptor directly interacts with the NLRP3 inflammasome scaffold protein

The P2X7 receptor (P2X7R) is a known and powerful activator of the NOD‐like receptor (NLR)P3 inflammasome; however, the underlying pathways are poorly understood. Thus, we investigated the molecular mechanisms involved. The effect of P2X7R expression and activation on NLRP3 expression and recruitment was investigated by Western blot, RT‐PCR, coimmunoprecipitation, and confocal microscopy in microglial mouse cell lines selected for reduced P2X7R expression and in primary cells from P2X7R‐/‐ C57BL/6 mice. We show here that P2X7R activation by ATP (EC50 = 1 mM) or benzoyl‐ATP (EC50 = 300 μM) and P2X7R down‐modulation caused a 2‐ to 8‐fold up‐regulation of NLRP3 mRNA in mouse N13 microglial cells. Moreover, NLRP3 mRNA was also up‐regulated in primary microglial and macrophage cells from P2X7R‐/‐ mice. Confocal microscopy and immunoprecipitation assays showed that P2X7R and NLRP3 closely interacted at discrete subplasmalemmal sites. Finally, P2X7R stimulation caused a transient (3‐4 min) cytoplasmic Ca2+ increase localized to small (2‐3 μm wide) discrete subplasmalemmal regions. The Ca2+ increase drove P2X7R recruitment and a 4‐fold increase in P2X7R/NLRP3 association within 1‐2 min. These data show a close P2X7R and NLRP3 interaction and highlight the role of P2X7R in the localized cytoplasmic ion changes responsible for both NLRP3 recruitment and activation.— Franceschini, A., Capece, M., Chiozzi, P., Falzoni, S., Sanz, J. M., Sarti, A. C., Bonora, M., Pinton, P., Di Virgilio, F. The P2X7 receptor directly interacts with the NLRP3 inflammasome scaffold protein. FASEB J. 29, 2450‐2461 (2015). www.fasebj.org

Purinoceptor function in the immune system

Immune cells express plasma membrane receptors for extracellular adenosine and purine and pyrimidine nucleotides. Overwhelming evidence suggests that these receptors have a pivotal role in the modulation of several immune cell responses, and may therefore be very important in the overall economy of the immune network. Adenosine can be either an activatory or an inhibitory mediator. Nucleotides have a more complex effect, depending not only on the P2 receptor subtype activated but also on doses applied and the length of stimulation. Both metabotropic (G‐protein‐linked) and ionotropic (intrinsic ion channels) P2 receptors are present in immune cells and their expression is modulated by inflammatory cytokines and differentiation agents. The physiological meaning of the P2 receptors is still obscure; however, converging evidence from different laboratories suggest that they may be involved in a wide variety of responses such proliferation, differentiation, cell death, and cytokine release. Drug Dev. Res. 39:319–329, 1996.

ATP receptors and giant cell formation.

We have investigated the role of the purinergic P2X7 receptor in the formation of multinucleated giant cells in human monocyte/macrophage cultures stimulated with either concanavalin A or phytohemagglutinin. Macrophage fusion can be blocked by a P2X7‐selective pharmacological antagonist or by a mAb directed against the extracellular P2X7 domain. Furthermore, macrophage cell clones expressing high P2X7 levels spontaneously fuse in culture, whereas macrophage clones lacking P2X7 are unable to fuse. Our findings suggest that the newly identified purinergic P2X7 receptor plays a central role in the complex chain of events leading to generation of macrophage‐derived giant cells. J. Leukoc. Biol. 66: 723–726; 1999.

Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR

BackgroundBreast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15–3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique.ResultsWe investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis.ConclusionsThe study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs.

PPARγ Pro12Ala and ACE ID polymorphisms are associated with BMI and fat distribution, but not metabolic syndrome

BackgroundMetabolic Syndrome (MetS) results from the combined effect of environmental and genetic factors. We investigated the possible association of peroxisome proliferator-activated receptor-γ2 (PPARγ2) Pro12Ala and Angiotensin Converting Enzyme (ACE) I/D polymorphisms with MetS and interaction between these genetic variants.MethodsThree hundred sixty four unrelated Caucasian subjects were enrolled. Waist circumference, blood pressure, and body mass index (BMI) were recorded. Body composition was estimated by impedance analysis; MetS was diagnosed by the NCEP-ATPIII criteria. A fasting blood sample was obtained for glucose, insulin, lipid profile determination, and DNA isolation for genotyping.ResultsThe prevalence of MetS did not differ across PPARγ2 or ACE polymorphisms. Carriers of PPARγ2 Ala allele had higher BMI and fat-mass but lower systolic blood pressure compared with Pro/Pro homozygotes. A significant PPARγ2 gene-gender interaction was observed in the modulation of BMI, fat mass, and blood pressure, with significant associations found in women only. A PPARγ2-ACE risk genotype combination for BMI and fat mass was found, with ACE DD/PPARγ2 Ala subjects having a higher BMI (p = 0.002) and Fat Mass (p = 0.002). Pro12Ala was independently associated with waist circumference independent of BMI and gender.ConclusionsCarriers of PPARγ2 Ala allele had higher BMI and fat-mass but not a worse metabolic profile, possibly because of a more favorable adipose tissue distribution. A gene interaction exists between Pro12Ala and ACE I/D on BMI and fat mass. Further studies are needed to assess the contribution of Pro12Ala polymorphism in adiposity distribution.