The Purinergic System as a Pharmacological Target for the Treatment of Immune-Mediated Inflammatory Diseases.

Immune-mediated inflammatory diseases (IMIDs) encompass a wide range of seemingly unrelated conditions, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases, asthma, chronic obstructive pulmonary disease, and systemic lupus erythematosus. Despite differing etiologies, these diseases share common inflammatory pathways, which lead to damage in primary target organs and frequently to a plethora of systemic effects as well. The purinergic signaling complex comprising extracellular nucleotides and nucleosides and their receptors, the P2 and P1 purinergic receptors, respectively, as well as catabolic enzymes and nucleoside transporters is a major regulatory system in the body. The purinergic signaling complex can regulate the development and course of IMIDs. Here we provide a comprehensive review on the role of purinergic signaling in controlling immunity, inflammation, and organ function in IMIDs. In addition, we discuss the possible therapeutic applications of drugs acting on purinergic pathways, which have been entering clinical development, to manage patients suffering from IMIDs.

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling.

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca2+ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca2+ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca2+ increase is specifically triggered by the autoreactive CD4+ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca2+ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.

[ROLE OF PURINERGIC RECEPTORS IN IMMUNE RESPONSE].

Purine receptors are located on immune and somatic cells of animal and human organisms. Summation of signals from purine and TOLL-like receptors takes place on the level of inflammasome formation and results in summation of the first and second signals of innate immunity. The first signal--from PAMPs (pathogen associated molecular patterns), the second--from DAMPs (danger associated molecular patterns). Adenosine triphosphate (ATP) is the most studied DAMP. ATP connects with purine receptors which include P2 (P2X7 receptors are the best described), that results in opening of channels of these receptors and transit of ATP into the cell. In parallel exit of K⁺ from cells and entrance of Ca²âº and Na⁺ into the cells is observed, that is associated with activation of the immune competent cell. Damaged cells dying via necrosis or apoptosis are the source of extracellular ATP, as well as activated immunocytes. Signals from P2 and TOLL-like receptors are summarized in effectors of immune response, and activation of P2 receptors in lymphocytes makes a contribution into activation of cells, mediated by T-cell receptor. Negative side of purine receptor activation is a stimulating effect on proliferation and metastasis of malignant cells. The practical output of knowledge on functioning of purine receptors for clinical immunology is the application of agonists and antagonists of purine receptors, as well as explanation of effect of immune modulators from the position of launch of K⁺/Na⁺-pump; resulting in prolonged activation of immune competent cells.

Purinergic receptor P2X7: a novel target for anti-inflammatory therapy.

Purinergic receptors, also known as purinoceptors, are ligand gated membrane ion channels involved in many cellular functions. Among all identified purinergic receptors, P2X7 subform is unique since it induces the caspase activity, cytokine secretion, and apoptosis. The distribution of P2X7 receptors, and the need of high concentration of ATP required to activate this receptor exhibited its ability to function as 'danger' sensor associated with tissue inflammation and damage. Further, the modulation of other signalling pathways associated with P2X7 has also been proposed to play an important role in the control of macrophage functions and inflammatory responses, especially towards lipopolysaccharides. Experimentally, researchers have also observed the decreased severity of inflammatory responses in P2X7 receptor expressing gene (P2RX7) knockout (KO) phenotypes. Therefore, newly developed potent antagonists of P2X7 receptor would serve as novel therapeutic agents to combat various inflammatory conditions. In this review article, we tried to explore various aspects of P2X7 receptors including therapeutic potential, and recent discoveries and developments of P2X7 receptor antagonists.

Purinergic receptors are required for HIV-1 infection of primary human macrophages.

Macrophages play a significant role in HIV infection, viral rebound, and the development of AIDS. However, the function of host proteins in viral replication is incompletely characterized in macrophages. Purinergic receptors P2X and P2Y are major components of the macrophage immune response to pathogens, inflammation, and cellular damage. We demonstrate that these receptors are necessary for HIV infection of primary human macrophages. Inhibition of purinergic receptors results in a significant reduction in HIV replication in macrophages. This inhibition is independent of viral strain and is dose dependent. We also identify that P2X(1), P2X(7), and P2Y(1) receptors are involved in viral replication. We show that P2X(1), but not P2X(7) or P2Y(1), is necessary for HIV entry into macrophages. We demonstrate that interaction of the HIV surface protein gp120 with macrophages stimulates an increase in ATP release. Thus, we propose that HIV's binding to macrophages triggers a local release of ATP that stimulates purinergic receptors and facilitates HIV entry and subsequent stages of viral replication. Our data implicate a novel role for a family of host proteins in HIV replication in macrophages and suggest new therapeutic targets to reduce the devastating consequences of HIV infection and AIDS.

Purinergic receptor type 6 contributes to airway inflammation and remodeling in experimental allergic airway inflammation.

RATIONALE: Extracellular nucleotides have recently been identified as proinflammatory mediators involved in asthma pathogenesis by signaling via purinergic receptors, but the role of the purinergic receptor type 6 (P2Y6R) has not been previously investigated. OBJECTIVES: To investigate the role of P2Y6R in asthma pathogenesis. METHODS: Acute and chronic OVA model and also HDM model of allergic inflammation in C57Bl/6 mice treated with specific P2Y6R antagonist and P2Y6R(-/-) mice were evaluated for classical features of asthmatic inflammation. In addition, primary epithelial cell culture from human and epithelial cell lines from mouse and human were stimulated with P2Y6R agonist and treated with P2Y6R antagonist and assessed for IL-6, IL-8/CXCL8 and KC levels. Experiments with P2Y6R(-/-) and P2Y6R(+/+) chimera were performed to discriminate the role of P2Y6R activation in structural lung cells and in cells from hematopoietic system. MEASUREMENTS AND MAIN RESULTS: We observed that the intratracheal application of a P2Y6R antagonist (MRS2578) and P2Y6R deficiency inhibited cardinal features of asthma, such as bronchoalveolar lavage eosinophilia, airway remodeling, Th2 cytokine production, and bronchial hyperresponsiveness in the ovalbumin-alum model. MRS2578 was also effective in reducing airway inflammation in a model using house dust mite extracts to induce allergic lung inflammation. Experiments with bone marrow chimeras revealed the importance of the P2Y6R expression on lung structural cells in airway inflammation. In accordance with this finding, we found a strong up-regulation of P2Y6 expression on airway epithelial cells of animals with experimental asthma. Concerning the underlying mechanism, we observed that MRS2578 inhibited the release of IL-6 and IL-8/KC by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6R agonist uridine-5'-diphosphate increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors induced the release of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro. CONCLUSIONS: P2Y6R expression on airway epithelial cells is up-regulated during acute and chronic allergic airway inflammation, and selective blocking of P2Y6R or P2Y6R deficiency on the structural cells reduces cardinal features of experimental asthma. Thus, blocking pulmonary P2Y6R might be a target for the treatment of allergic airway inflammation.

The Complexity of Purinergic Signaling During Toxoplasma Infection.

Toxoplasmosis is a neglected disease caused by infection by the protozoan Toxoplasma gondii. One-third of the global population is expected to be by infected T. gondii. In Europe and North America, most infections do not induce disease, except in the context of immunosuppression. However, in endemic regions such Central and South America, infections induce severe ocular and potentially lethal disease, even in immunocompetent individuals. The immune response against T. gondii infection involves components of innate immunity even in the chronic phase of the disease, including dangerous signal molecules such as extracellular nucleotides. Purinergic signaling pathways include ionotropic and metabotropic receptors activated by extracellular nucleotides that are divided into P2X, P2Y, and A1 receptor families. The activation of purinergic signaling impacts biological systems by modulating immune responses to intracellular pathogens such as T. gondii. Ten years ago, purinergic signaling in the T. gondii infection was reported for the first time. In this review, we update and summarize the main findings regarding the role of purinergic signaling in T. gondii infection; these include in vitro findings: the microbicidal effect of P2Y and P2X7 activation phagocytic cells and parasite control by P2X7 activation in non-phagocytic cells; and in vivo findings: the promotion of early pro-inflammatory events that protect the host in acute and chronic models.

Unveiling the Potential of Purinergic Signaling in Schistosomiasis Treatment.

Schistosomiasis is a neglected tropical disease. It is related to long-lasting granulomatous fibrosis and inflammation of target organs, and current sub-optimal pharmacological treatment creates global public health concerns. Intravascular worms and eggs release antigens and extracellular vesicles that target host endothelial cells, modulate the immune system, and stimulate the release of damageassociated molecular patterns (DAMPs). ATP, one of the most studied DAMPs, triggers a cascade of autocrine and paracrine actions through purinergic P2X and P2Y receptors, which are shaped by ectonucleotidases (CD39). Both P2 receptor families, and in particular P2Y1, P2Y2, P2Y12, and P2X7 receptors, have been attracting increasing interest in several inflammatory diseases and drug development. Current data obtained from the murine model unveiled a CD39-ADP-P2Y1/P2Y12 receptors signaling pathway linked to the liver and mesenteric exacerbations of schistosomal inflammation. Therefore, we proposed that members of this purinergic signaling could be putative pharmacological targets to reduce schistosomal morbidity.

Role of purinergic system and vitamin D in the anti-cancer immune response.

For several years, scientists have recognized that vitamin D plays an important role in mineral and bone homeostasis. It was mostly used to treat osteoporosis and rickets in the past decades. Vitamin D has also been discovered to be modulator of the immune system and may play a role in a variety of diseases, including autoimmune diseases, in recent years. Vitamin D interaction with the vitamin D receptor (VDR), which has transcriptional imparts and is displayed on a variety of cell types, including those of the immune system, appears to be accountable for the immune-modulating effects. The action of tumor cells and vitamin D were the first to be investigated, but the spotlight is now on immunologic and purinergic systems. We conducted a systematic search in Pub Med as well as Google scholar for studies written in English. Vitamin D, cancer, purinergic signaling, and immune response were among the search words. Vitamin D has the potential to be a useful coadjuvant in cancer therapy and the purinergic system may be a potential treatment target to cancer therapy, according to our findings.

Purinergic signalling in host innate immune defence against intracellular pathogens.

Purinergic signalling is an evolutionarily conserved signalling pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides released from host cells during intracellular pathogen infections activate plasma membrane purinergic type 2 receptors (P2 receptors) that stimulate microbicidal mechanisms in host innate immune cells. P2X ion channels and P2Y G protein-coupled receptors are involved in activating host innate immune defence mechanisms, phagocytosis, phagolysosomal fusion, production of reactive species, acidification of parasitophorous vacuoles, inflammasome activation, and the release of cytokines, chemokines, and other inflammatory mediators. In this review, as part of a special issue in tribute to Geoffrey Burnstock, we discuss advances in understanding the importance of P2 receptors in the host antimicrobial innate mechanisms against intracellular pathogen infections.

Purinergic signaling: a novel mechanism in immune surveillance.

Purinergic receptors and the associated signaling cascades are known to play critical roles in cardiovascular, nervous, respiratory, gastrointestinal and urinogenital systems. Recent studies have also shed light on the importance of nucleotides and purinergic receptors in the regulation of the immune response. With a better understanding of the distribution and the receptor subtypes, the purinoceptors have the potential to become important therapeutic targets in inflammation, chemotaxis and immune-related diseases.

Eosinophils and Purinergic Signaling in Health and Disease.

Eosinophils are major effector cells against parasites, fungi, bacteria, and viruses. However, these cells also take part in local and systemic inflammation, which are central to eczema, atopy, rhinitis, asthma, and autoimmune diseases. A role for eosinophils has been also shown in vascular thrombotic disorders and in cancer. Many, if not all, above-mentioned conditions involve the release of intracellular nucleotides (ATP, ADP, UTP, etc.) and nucleosides (adenosine) in the extracellular environment. Simultaneously, eosinophils further release ATP, which in autocrine and paracrine manners, stimulates P2 receptors. Purinergic signaling in eosinophils mediates a variety of responses including CD11b induction, ROI production, release of granule contents and enzymes, as well as cytokines. Exposure to extracellular ATP also modulates the expression of endothelial adhesion molecules, thereby favoring eosinophil extravasation and accumulation. In addition, eosinophils express the immunosuppressive adenosine P1 receptors, which regulate degranulation and migration. However, pro-inflammatory responses induced by extracellular ATP predominate. Due to their important role in innate immunity and tissue damage, pharmacological targeting of nucleotide- and nucleoside-mediated signaling in eosinophils could represent a novel approach to alleviate eosinophilic acute and chronic inflammatory diseases. These innovative approaches might also have salutary effects, particularly in host defense against parasites and in cancer.

Purinergic receptors and the inflammatory response mediated by lipids.

The inflammatory response is regulated by the production of different extracellular mediators, including lipids and extracellular nucleotides. In the extracellular environment, intermediate lipids activate specific G-protein-coupled receptors (GPCRs) in target cells and promote cell recruitment and activation. Extracellular nucleotides activate two types of receptors, the ionotropic purinergic P2X and the metabotropic purinergic P2Y receptors, inducing the release of cytokines and promoting cell recruitment. Several P2X receptors are associated with an increase in the production of immunoactive lipids mediators, which in turn are able to interfere with the activation of different P2Y receptors, establishing a tight signalling link between purinergic receptors and lipid mediators. In this review, we summarise recent studies indicating signalling crosstalk between purinergic P2X and P2Y receptor activation and lipid mediators with a focus on inflammatory diseases. Novel concepts arising from this crosstalk would result in the development of combinatorial therapies targeting lipid synthesis together with individual P2 receptors for the management of inflammatory diseases.

Changes in phagocytosis and potassium channel activity in microglia of 5xFAD mice indicate alterations in purinergic signaling in a mouse model of Alzheimer's disease.

As the immunocompetent cells of the central nervous system, microglia accumulate at amyloid beta plaques in Alzheimer's disease (AD) and acquire a morphological phenotype of activated microglia. Recent functional studies, however, indicate that in mouse models of amyloidosis and AD, these cells are rather dysfunctional indicated by a reduced phagocytic activity. Here, we report that this reduction in phagocytic activity is associated with perturbed purinergic receptor signaling, since phagocytosis could be stimulated by P2Y6 receptor activation in control, but not in 5xFAD transgenic animals, an animal model of amyloid deposition. Impaired phagocytosis is not innate, and develops only at later stages of amyloidosis. Furthermore, we show that membrane currents induced by uridine diphosphate, a ligand activating P2Y6 receptors, are altered in response rate and amplitude in microglia in close vicinity to plaques, but not in plaque-free areas of 5xFAD animals. These changes were accompanied by changes in membrane properties and potassium channel activity of plaque-associated microglia in early and late stages of amyloidosis. As a conclusion, the physiological properties of plaque-associated microglia are altered with a strong impact on purinergic signaling.

Purinergic receptors modulate MAP kinases and transcription factors that control microglial inflammatory gene expression.

Following many types of brain injury, microglial cell hyperactivation, and the subsequent release of neurotoxic mediators into the CNS contributes to inflammation and neuronal death. Among the proteins important for modulating the inflammatory function of microglia are the P2 purinergic receptors for which extracellular adenine nucleotides, such as ATP, are ligands. Because adenine nucleotides are abundant in the extracellular fluid following brain injury, ATP may represent an important component of the inflammatory microenvironment controlling microglial cell function. Although much work has been done examining the mechanisms whereby adenine nucleotides stimulate inflammatory mediator production, little is known concerning their complementary inhibitory effects. In this review we will focus on what is currently known about the microglial inhibitory effects of adenine nucleotides in the context of inflammation and summarize the current knowledge of their effects via purinergic receptors on microglial signal transduction pathways including transcription factors important for controlling inflammatory gene expression. The relevance of these mechanisms to microglial inflammatory function and physiology will be discussed. Further, we present data here illustrating that MAP kinase signal transduction pathways are altered in activated microglia that have been primed with or co-exposed to adenine nucleotides; effects that are stimulus- and MAPK pathway-specific. We also demonstrate the ability of P2X7 receptors to stimulate the phosphorylation of CREB, a putative inhibitory transcription factor in microglia. Together, these data indicate that ATP may be an endogenous inhibitor or neuroprotective molecule decreasing the inflammatory capacity of microglia.

Purinergic Signaling and the Immune Response in Sepsis: A Review.

PURPOSE: Sepsis remains an unresolved clinical problem with high in-hospital mortality. Despite intensive research over decades, no treatments for sepsis have become available. Here we explore the role of ATP in the pathophysiology of sepsis. ATP is not only a universal energy carrier but it also acts as an extracellular signaling molecule that regulates immune function. ATP stimulates a large family of purinergic receptors found on the cell surface of virtually all mammalian cells. In severe sepsis and septic shock, ATP is released in large amounts into the extracellular space where it acts as a "danger" signal. In this review, we focus on the roles of ATP as a key regulator of immune cell function and as a disruptive signal that contributes to immune dysfunction in sepsis. METHODS: We summarized the current understanding of the pathophysiology of sepsis, with special emphasis on the emerging role of systemic ATP as a disruptive force that promotes morbidity and mortality in sepsis. FINDINGS: Over the past two decades, the discovery that regulated ATP release and purinergic signaling represent a novel regulatory mechanism in immune cell physiology has opened up new possibilities in the treatment of sepsis. Immune cells respond to stimulation with the release of cellular ATP, which regulates cell functions in autocrine and paracrine fashions. In sepsis, large amounts of systemic ATP produced by tissue damage and inflammation disrupt these regulatory purinergic signaling mechanisms, leading to immune dysfunction that promotes the pathophysiologic processes involved in sepsis. IMPLICATIONS: The knowledge of these ATP-dependent signaling processes is likely to reveal exciting new avenues in the treatment of the unresolved clinical problem of sepsis.

Immunosuppressive activities of adenosine in cancer.

Multiple immunosuppressive mechanisms impede anti-tumor immunity. Among them, the accumulation of extracellular adenosine is a potent and widespread strategy exploited by tumors to escape immunosurveillance through the activation of purinergic receptors. In the immune system, engagement of A2a and A2b adenosine receptors is a critical regulatory mechanism that protects tissues against excessive immune reactions. In tumors, this pathway is hijacked and hinders anti-tumor immunity, promoting cancer progression. Different groups have highlighted the therapeutic potential of blocking CD73-dependent adenosine-mediated immunosuppression to reinstate anti-tumor immunity. Phase clinical trials evaluating anti-CD73 antibodies and A2a receptor antagonists in cancer patients are currently ongoing. We here review the recent literature on the immunosuppressive effects of extracellular adenosine and discuss the development of adenosine inhibitors.

Development of an antiserum to rat-brain A1 adenosine receptor: application for immunological and structural comparison of A1 adenosine receptors from various tissues and species.

An antiserum was developed in a rabbit against rat-brain A1 adenosine receptor. This antiserum recognized the denatured form of the purified rat-brain A1 adenosine receptor in immunoblot analysis and the native form of the receptor in the immunoprecipitation analysis. Immunoblot analysis of unpurified or purified adenosine receptor preparations from rat-brain membranes revealed a major immunoreactive band at a position of molecular mass of approx. 35 kDa, which corresponds to the position of purified rat-brain A1 adenosine receptor. Although A1 adenosine receptors from other rat tissues such as testis and adipocyte were also found to be immunoreactive with this antiserum by immunoblot analysis, purified human-brain A1 adenosine receptors showed a poor reactivity with this antibody. The order of the relative immunoreactivity of these A1 adenosine receptors with the antiserum was found to be brain > adipocyte > or = testis. Moreover, the immunoreactivity of these receptors significantly increased after these receptor preparations were deglycosylated by endoglycosidase F. After the deglycosylation, no significant differences in both the immunoreactivity and molecular mass among these receptor preparations were found on the immunoblot. These results suggest that the differences in the molecular mass or immunoreactivity among the A1 adenosine receptor preparations from three rat tissues were mainly due to the difference of sugar moiety present in each receptor molecule. These data are the first to provide analyses of immunological characteristics of A1 adenosine receptors from different tissues and species.

Asthma: the interplay between viral infections and allergic diseases.

Respiratory viruses and allergens synergistically contribute to disease pathogenesis in asthma. Potential mechanisms underlying this clinically relevant association are the subject of intense investigation. This review summarizes current knowledge and recent advances in this area, with an emphasis on potential mechanisms involving immunoglobulin E, type I interferon antiviral responses, epithelial factors, and the role of dendritic cells and other antigen-presenting cells in linking viral and allergic inflammatory responses relevant to asthmatic disease.

Role of anti-calcium channel and anti-receptor autoantibodies in autonomic dysfunction in Sjögren's syndrome.

Auto-antibodies cross-reacting with L-type voltage-gated calcium channels (VGCCs) have been described in primary Sjögren's syndrome (pSS), and may mediate the cardiac defects in neonates born to mothers with pSS. L-type VGCCs are also present in autonomically innervated tissues. Therefore, the aim of this project was to investigate a role for anti-VGCC antibodies and antibodies to alpha(1)-adrenoceptors or P(2X)-purinoceptors in the autonomic dysfunction that occurs in pSS. Contraction of the sympathetically innervated vas deferens in response to stimulation of the muscle by an alpha(1)-adrenoceptor agonist (phenylephrine) or a P(2X)-purinoceptor agonist (alpha,beta-methylene ATP) was measured in the absence and presence of 2% serum. Contractions produced by phenylephrine and by alpha,beta-methylene ATP were abolished by nicardipine, demonstrating that they are coupled to calcium influx through L-type VGCCs. Serum from patients with pSS or from healthy controls did not significantly alter the L-type channel-dependent responses of smooth muscle to agonist stimulation. We therefore conclude that pSS serum does not contain autoantibodies that functionally inhibit L-type VGCCs, alpha(1)-adrenoceptors or P(2X)-purinoceptors in smooth muscle and that such autoantibodies cannot explain the autonomic dysfunction in pSS.