Francesco Di Virgilio

The P2X7 Receptor: A Key Player in IL-1 Processing and Release

Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X7 receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X7 receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.

Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice

The release of adenosine triphosphate through autophagy can promote antitumor immune responses. Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. Here we demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, reestablished the recruitment of immune cells, and restored chemotherapeutic responses but only in immunocompetent hosts. Thus, autophagy is essential for the immunogenic release of ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.

The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide‐induced apoptosis: significance for the molecular mechanism of Bcl‐2 action

The mechanism of action of the anti‐apoptotic oncogene Bcl‐2 is still largely obscure. We have recently shown that the overexpression of Bcl‐2 in HeLa cells reduces the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) by increasing the passive Ca2+ leak from the organelle. To investigate whether this Ca2+ depletion is part of the mechanism of action of Bcl‐2, we mimicked the Bcl‐2 effect on [Ca2+]er by different pharmacological and molecular approaches. All conditions that lowered [Ca2+]er protected HeLa cells from ceramide, a Bcl‐2‐sensitive apoptotic stimulus, while treatments that increased [Ca2+]er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca2+ from the endoplasmic reticulum and thus [Ca2+] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca2+]er levels, as well as the buffering of cytoplasmic [Ca2+] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl‐2‐dependent reduction of [Ca2+]er is an important component of the anti‐apoptotic program controlled by this oncogene.

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial hyper-reactivity, were abrogated when lung ATP levels were locally neutralized using apyrase or when mice were treated with broad-spectrum P2-receptor antagonists. In addition to these effects of ATP in established inflammation, Th2 sensitization to inhaled antigen was enhanced by endogenous or exogenous ATP. The adjuvant effects of ATP were due to the recruitment and activation of lung myeloid dendritic cells that induced Th2 responses in the mediastinal nodes. Together these data show that purinergic signaling has a key role in allergen-driven lung inflammation that is likely to be amenable to therapeutic intervention.

Calcium and apoptosis: facts and hypotheses

Although longstanding experimental evidence has associated alterations of calcium homeostasis to cell death, only in the past few years the role of calcium in the signaling of apoptosis has been extensively investigated. In this review, we will summarize the current knowledge, focusing on (i) the effect of the proteins of the Bcl-2 family on ER Ca2+ levels, (ii) the action of the proteolytic enzymes of apoptosis on the Ca2+ signaling machinery, (iii) the ensuing alterations on the signaling patterns of extracellular stimuli, and (iv) the intracellular targets of ‘apoptotic’ Ca2+ signals, with special emphasis on the mitochondria and cytosolic Ca2+-dependent enzymes.

The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death

Many immune and inflammatory cells express a plasma membrane receptor for extracellular ATP, termed the P2Z purinoceptor, which appears to be coupled to a plasma membrane pore. The physiological role of such a molecule is generally unknown, except for the striking susceptibility to ATP-mediated cytotoxicity that it confers. The receptor is upregulated in human monocytes by interferon gamma and is also expressed during macrophage differentiation. Here, Francesco Di Virgilio discusses recent information on this receptor, and suggests a possible role for it in the immune and inflammatory response.

Increased level of extracellular ATP at tumor sites: In vivo imaging with plasma membrane luciferase

Background There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP. Methodology/Principal Findings Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours. Conclusions/Significance Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

ATP-mediated cytotoxicity in microglial cells.

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop.

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X7R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X7R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X7R blockade or genetic deficiency of P2X7R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X7R could lead to the development of tolerance without the need for intensive immunosuppression.

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.