Elevation Of Extracellular Adenosine Research Articles

Muscle satellite cells are impaired in type 2 diabetic mice by elevated extracellular adenosine.

Muscle regeneration is known to be defective under diabetic conditions. However, the underlying mechanisms remain less clear. Adult quiescent muscle satellite cells (MuSCs) from leptin-receptor-deficient (i.e., db/db) diabetic mice are defective in early activation invivo, but not in culture, suggesting the involvement of pathogenic niche factors. Elevated extracellular adenosine (eAdo) and AMP (eAMP) are detected under diabetic conditions. eAdo and eAMP potently inhibit cell cycle re-entry of quiescent MuSCs and injury-induced muscle regeneration. Mechanistically, eAdo and eAMP engage the equilibrative Ado transporters (ENTs)-Ado kinase (ADK)-AMPK signaling axis in MuSCs to inhibit the mTORC1-dependent cell growth checkpoint. eAdo and eAMP also inhibit early activation of quiescent fibroadipogenic progenitors and human MuSCs by the same mechanism. Treatment of db/db diabetic mice with an ADK inhibitor partially rescues the activation defects of MuSCs invivo. Thus, both ADK and ENTs represent potential therapeutic targets for restoring the regenerative functions of tissue stem cells in patients with diabetes.

Adenosine Signaling Perturbs Erythropoiesis and Promotes Myeloid Differentiation

BackgroundAdenosine is a major signaling nucleoside that activates cellular signaling pathways through a family of four different G protein-coupled adenosine receptors (ARs), A 1, A 2A, A 2B, and A 3. At steady state conditions, extracellular levels of adenosine remain low (10 to 200 nM) either through its rapid cellular uptake by specialized nucleoside transporters, mainly through the equilibrative nucleoside transporter 1 (ENT1), or its degradation by adenosine deaminases. However, the extracellular levels of adenosine can be rapidly elevated up to 100 μM in response to hypoxia, inflammation, or tissue injury. Under pathophysiological conditions, adenosine signaling is involved in modulating inflammation, fibrosis, and ischemic tissue injury. In sickle cell disease (SCD), adenosine signaling is enhanced and contributes to the pathophysiology of the disease. Despite the importance of adenosine signaling in regulating cell proliferation, and stem cell regeneration, as well as in red blood cell functions and adaptation to hypoxia, very little is known about its implication in hematopoiesis, and more specifically during erythropoiesis. Here, we aimed to investigate the effects of high extracellular adenosine on the erythroid commitment and differentiation of hematopoietic progenitors, and to decipher the implication of ARs in these processes.ResultsTo investigate the role of high extracellular adenosine in regulating erythroid commitment and differentiation of hematopoietic progenitors, we performed ex vivo erythropoiesis of healthy CD34 + cells in the presence or absence of increased extracellular adenosine concentrations ranging from 10 to 200 µM. Our results showed that adenosine decreases erythroid proliferation in a dose dependent manner. High adenosine levels (>50μM) inhibited the proliferation of erythroid precursors and increased apoptosis via a cell cycle arrest in G1. Accordingly, western blots revealed the accumulation of p53 and its downstream target p21, a well-known mediator of G1 cell-cycle arrest, in adenosine-treated cells.Moreover, adenosine treatment led to the persistence of a non-erythroid GPA neg subpopulation expressing myeloid markers (CD18, CD11a, CD13, CD33). May-Grünwald Giemsa staining of this subset revealed granular cells at different stages of differentiation. The culture of FACS-sorted CD36 + and CD36 - cells suggested that this adenosine-induced GPA neg population originates from the survival of CD36 - myeloid progenitors even in the presence of erythropoietin. Importantly, these effects were specific to adenosine as neither guanosine, uridine nor cytidine affected the proliferation and differentiation of erythroid precursors. Furthermore, we have recently shown that ENT1-mediated adenosine uptake is essential for optimal erythroid differentiation. Therefore, we suggested that elevated extracellular adenosine perturbs erythropoiesis via its signaling upon ARs activation. To confirm this hypothesis, we assessed the effect of ARs activation during erythropoiesis. Given that A 2B and A 3 are the only known ARs expressed in human hematopoietic progenitors and erythroid precursors, we used BAY60-6583 and CI-IB-MECA, two highly selective agonists for A 2B and A 3 receptors, respectively. Both BAY60-6583 and CI-IB-MECA increased apoptosis and decreased erythroblast maturation and enucleation, while only Cl-IB-MECA led to the upregulation of CD33 and CD11a myeloid markers and promoted the differentiation of a GPA neg myeloid subpopulation.ConclusionOverall, our results place adenosine signaling as a new player in hematopoiesis regulation. Adenosine signaling via A 3 perturbs erythropoiesis and promotes the survival and differentiation of myeloid progenitors even in an erythroid favoring environment. While the activation of A 2B hampers optimal erythropoiesis without impacting the myeloid differentiation. As both ineffective erythropoiesis and increased leucocyte counts are reported in SCD, and given the detrimental role of high adenosine levels in its pathophysiology, further studies are ongoing to address the impact of adenosine signaling on hematopoiesis in this disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Abstract 1683: A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity

Abstract High levels of extracellular adenosine in the tumor microenvironment are known to play a significant role in tumor immune evasion and promote tumor growth and metastasis. We defined the receptor(s) required for mediating the effect of adenosine on immune cells within the tumor microenvionment and report the characterization of a novel Immuno-Oncology-dedicated adenosine receptor 2A antagonist that functions in the high adenosine concentration found in tumors. We first explored the expression of the four adenosine receptors in primary human immune cells. A2A receptor was the main adenosine receptor expressed by CD4 and CD8 T lymphocytes and monocytes, and the only one in mature monocyte-derived dendritic cells and NK cells. A2B receptor was poorly detected in T cells and monocytes, while A1 and A3 receptors were never detected. Given these expression patterns, we further studied A2A functions in primary human T lymphocytes and monocytes. Selective A2A agonists such as CGS-21680 strongly suppressed cytokine production by activated primary human T lymphocytes, thus highlighting that A2A is the main effector receptor of the sensing of adenosine in tumors. We further confirmed the elevated extracellular adenosine level in the tumor microenvironment in several mouse and human tumors. High adenosine levels correlated with strong tumoral expression of CD73, the enzyme that converts AMP to adenosine. Interestingly, we showed that A2A receptor antagonists designed for Parkinson’s disease dramatically lost potency in a high adenosine environment ; our data indicated that a 30-fold dose increase may be required for full target inhibition within tumors. Therefore we developed a novel and potent A2A blocker with sub-nanomolar Ki and IC50 in a cAMP assay and a more than 100-fold selectivity over other adenosine receptors. Our lead compound kept a high potency in an adenosine-rich environment and restored cytokine production even in the presence of high concentrations of A2A agonists. iTeos inhibitor also efficiently reversed AMP-mediated T cell suppression. Furthermore, our compound rescued A2A receptor agonist-induced decrease of TNFα production by primary human monocytes, and was able to potently increase CD8 T cell cytotoxicity in a cytotoxicity assay with CD8 T cells as effectors and cancer cells as targets. These results suggest that iTeos new generation of A2A receptor antagonist, designed to keep a high potency in the adenosine-rich tumor microenvironment, may offer a new therapeutic opportunity in Immuno-Oncology. Citation Format: Erica Houthuys, Margreet Brouwer, Florence Nyawouame, Romain Pirson, Reece Marillier, Theo Deregnaucourt, Joao Marchante, Jakub Swiercz, Charlotte Moulin, Vanesa Bol, Gregory Driessens, Michel Detheux, Christophe Quéva, Stefano Crosignani, Bruno Gomes. A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2017-1683

Molecular mechanisms for adenosine regulation of helper T cell activation

Abstract Polarization of T helper subsets into different functional phenotypes strongly affects the progression of immune-related diseases. One of the hallmarks of immune cell activation and inflammation is the elevation of extracellular adenosine and increased expression of adenosine A2A receptors in activated immune cells. Adenosine strongly inhibits activation of helper T cells by elevating cAMP concentrations, which activate PKA and EPAC proteins to regulate cellular responses. Here we showed that adenosine strongly inhibits T cell accumulation rather than differentiating into functional subsets in the presence of polarizing conditions. Different aspects of adenosine mediated T cell suppression were phenocopied by specific activation of PKA and/or EPAC pathways. Adenosine can suppress phospho-activation of Akt pathway to promote T cell quiescence and to inhibit immediate downstream events after TCR stimulation. One of the targets for Akt is Foxo1. Foxo1 is known to suppress T cell proliferation. Our results showed that adenosine receptor stimulation reduced inhibitory phosphorylation of Foxo1 downstream of Akt. Foxo1 inhibitor, AS1842856, completely reversed the inhibition of T cell accumulation by adenosine signaling. Our results suggest that adenosine by activating PKA and/or EPAC pathways and by sustaining Foxo1 activation suppresses T cell activation and accumulation. These findings have important implications to develop novel interventions to regulate helper T cell responses in different pathological conditions.

Ischemic preconditioning with a ketogenic diet improves brain ischemic tolerance through increased extracellular adenosine levels and hypoxia-inducible factors

Ischemic tolerance reduces brain damage and neurological dysfunction after brain ischemia. A ketogenic diet (KD) has disease-modifying effects in several neurodegenerative disorders. In this study, we fed mice with a KD for three weeks and performed reversible middle cerebral artery occlusion (MCAO) in the animals. KD-fed mice had a significantly reduced infarct volume, increased regional cerebral blood flow (rCBF) and extracellular adenosine levels in both the ischemic and the reperfusion phases. In vitro and in vivo experiments revealed that the KD-induced neuroprotection was mediated through the adenosine A1 receptor. The KD increased Akt and ERK1/2 phosphorylation via A1R activation. Besides, the KD also upregulated robustly HIF-1α/HIF-2α and HIF regulated genes, such as VEGF and EPO. A three-week preconditioning period with a KD improved ischemic tolerance in mice with MCAO. The underlying mechanisms might include elevated extracellular adenosine levels, and increased Akt and ERK1/2 phosphorylation via A1 adenosine receptor activation, together with upregulated HIFs and HIF-regulated genes.

The adenosine generating enzymes CD39/CD73 control microglial processes ramification in the mouse brain.

Microglial cells invade the brain as amoeboid precursors and acquire a highly ramified morphology in the postnatal brain. Microglia express all essential purinergic elements such as receptors, nucleoside transporters and ecto-enzymes, including CD39 (NTPDase1) and CD73 (5'-nucleotidase), which sequentially degrade extracellular ATP to adenosine. Here, we show that constitutive deletion of CD39 and CD73 or both caused an inhibition of the microglia ramified phenotype in the brain with a reduction in the length of processes, branching frequency and number of intersections with Sholl spheres. In vitro, unlike wild-type microglia, cd39-/- and cd73-/- microglial cells were less complex and did not respond to ATP with the transformation into a more ramified phenotype. In acute brain slices, wild-type microglia retracted approximately 50% of their processes within 15 min after slicing of the brain, and this phenomenon was augmented in cd39-/- mice; moreover, the elongation of microglial processes towards the source of ATP or towards a laser lesion was observed only in wild-type but not in cd39-/- microglia. An elevation of extracellular adenosine 1) by the inhibition of adenosine transport with dipyridamole, 2) by application of exogenous adenosine or 3) by degradation of endogenous ATP/ADP with apyrase enhanced spontaneous and ATP-induced ramification of cd39-/- microglia in acute brain slices and facilitated the transformation of cd39-/- and cd73-/- microglia into a ramified process-bearing phenotype in vitro. These data indicate that under normal physiological conditions, CD39 and CD73 nucleotidases together with equilibrative nucleoside transporter 1 (ENT1) control the fate of extracellular adenosine and thereby the ramification of microglial processes.

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase, and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

Peroxynitrite treatment reduces adenosine uptake via the equilibrative nucleoside transporter in rat astrocytes

In the oxidative stress-loaded brain, extracellular adenosine levels are elevated and thereby neuronal damage is attenuated, but mechanisms underlying alteration of the extracellular kinetics of adenosine remain unclear. Here we investigated whether oxidative stress might alter functional expression of nucleoside transporters (NTs), a predominant regulatory system for nucleoside kinetics, in cultured rat astrocytes. Treatment of astrocytes with 0.5mM SIN-1 for 3h caused apparent cellular accumulation of nitrotyrosine, but had no effect on their viability, indicating load of oxidative stress to astrocytes without any change in their viability. Under the condition, [3H]adenosine uptake was significantly less than that by control cells. This decreased uptake was due to decrease in adenosine uptake mediated by an equilibrative NT (ENT) 1 which was inhibited by low concentrations (≤0.1μM) of nitrobenzylthioinosine (NBMPR), but not by sodium-dependent or high concentrations (≥1μM) of NBMPR-inhibitable nucleoside transporters. The expression level of ENT1 was not altered, while the Michaelis constant, but not the maximum rate, of adenosine uptake was increased. These findings suggest that under oxidative stress-loaded conditions, decreased adenosine clearance via astrocytic ENT1 might involve, at least in part, in an elevated extracellular adenosine level in the brain.

The Role of Adenosine Receptor Agonists in Regulation of Hematopoiesis

The review summarizes data evaluating the role of adenosine receptor signaling in murine hematopoietic functions. The studies carried out utilized either non-selective activation of adenosine receptors induced by elevation of extracellular adenosine or by administration of synthetic adenosine analogs having various proportions of selectivity for a particular receptor. Numerous studies have described stimulatory effects of non-selective activation of adenosine receptors, manifested as enhancement of proliferation of cells at various levels of the hematopoietic hierarchy. Subsequent experimental approaches, considering the hematopoiesis-modulating action of adenosine receptor agonists with a high level of selectivity to individual adenosine receptor subtypes, have revealed differential effects of various adenosine analogs. Whereas selective activation of A1 receptors has resulted in suppression of proliferation of hematopoietic progenitor and precursor cells, that of A3 receptors has led to stimulated cell proliferation in these cell compartments. Thus, A1 and A3 receptors have been found to play a homeostatic role in suppressed and regenerating hematopoiesis. Selective activation of adenosine A3 receptors has been found to act curatively under conditions of drug- and radiation-induced myelosuppression. The findings in these and further research areas will be summarized and mechanisms of hematopoiesis-modulating action of adenosine receptor agonists will be discussed.

Stimulation of ecto-5′-nucleotidase in human umbilical vein endothelial cells by lipopolysaccharide

The involvement of ecto-5'-nucleotidase (E-5'Nu) in the elevation of extracellular adenosine during inflammation is unclear. In the present study, the effect of lipopolysaccharide (LPS), an inflammation inducer, was investigated on E-5'Nu in human umbilical vein endothelial cells (HUVECs). E-5'Nu activity was enhanced after a 24 h exposure to LPS. This effect was dose dependent, with an EC50 of 1.66 ng/ml. At 10 microM, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 abolished the LPS-induced E-5'Nu activity. However, at 10 microM, the NF-kappaB inhibitor ammonium pyrrolidine dithiocarbamate had no effect. LPS upregulated the protein expression but not the messenger RNA expression of E-5'Nu. The inhibition of E-5'Nu by 100 microM alpha,beta-methylene adenosine-5'-diphosphate increased the LPS-induced inflammation, suggesting that E-5'Nu plays a significant role in reducing inflammation, probably through the generation of adenosine. In conclusion, the experiments indicate that LPS upregulates E-5'Nu activity in HUVECs through a PI3K-dependent increase in the abundance of E-5'Nu on cell membranes. Since adenosine is an anti-inflammatory molecule, E-5'Nu upregulation may be crucial in protecting endothelial cells against inflammatory damage.

Adenosinergic Regulation of Sleep: Multiple Sites of Action in the Brain

Adenosinergic Regulation of Sleep: Multiple Sites of Action in the Brain

NMDA receptor-mediated extracellular adenosine accumulation in rat forebrain neurons in culture is associated with inhibition of adenosine kinase.

The effect of N-methyl-d-aspartate (NMDA) on regulation of extracellular adenosine was investigated in rat forebrain neurons in culture. NMDA evoked accumulation of extracellular adenosine with an EC50 value of 4.8 +/- 1.2 microM. The effect of NMDA was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5,10-imine hydrogen maleate indicating that NMDA receptor activation was involved. The NMDA effect was also blocked by chelation of extracellular Ca2+ indicating that influx of calcium was required. The nitric oxide-cyclic GMP signalling pathway was not involved, as nitric oxide synthase inhibitors were unable to block, and cGMP analogs were unable to mimic, the effect of NMDA. The source for extracellular adenosine was likely to be intracellular adenosine as the ecto-5'-nucleotidase inhibitor alpha beta-methylene-ADP was unable to block the effect of NMDA. One possible cause of intracellular adenosine accumulation might be NMDA receptor-mediated inhibition of mitochondrial function and ATP hydrolysis. We found that NMDA caused a concentration dependent depletion of intracellular ATP with an EC50 value of 21 +/- 8 microM. NMDA also caused a significant decrease in adenosine kinase activity, assayed by two different methods. Consistent with the hypothesis that inhibition of adenosine kinase is sufficient to cause an increase in extracellular adenosine, inhibition of adenosine kinase by 5'-iodotubercidin resulted in elevation of extracellular adenosine. However, in the presence of a concentration of 5'-iodotubercidin that inhibited over 90% of adenosine kinase activity, exposure to NMDA still caused adenosine accumulation. These studies suggest that several possible mechanisms are likely to be involved in NMDA-evoked extracellular adenosine accumulation.

Influence of the joint treatment with granulocyte colony-stimulating factor and drugs elevating extracellular adenosine on erythropoietic recovery following 5-fluorouracil-induced haematotoxicity in mice.

The presented data address the problem of pleiotropic effects of granulocyte colony-stimulating factor (G-CSF) and suggest the ability of drugs increasing the level of extracellular adenosine to activate erythropoiesis when given jointly with G-CSF. To demonstrate these interactions, the effects of the drugs on the recovery from erythropoietic damage induced in mice by a single dose of 5-fluorouracil (5-FU) were investigated. Elevation of extracellular adenosine and thus activation of adenosine receptors was induced by joint administration of dipyridamole (DP), a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The drugs were injected in a 4-d treatment regimen starting 2 h after 5-FU injection. Both DP+AMP and G-CSF alone induced only weak effects. However, the combination of the three drugs produced significant elevation of erythrocytes in the peripheral blood which pertained in the posttreatment period. Stimulation of proliferation of erythroid progenitor cells (BFU-E) in femoral bone marrow and increased levels of reticulocytes in the peripheral blood were observed in the course of the 4-d treatment regimen. In addition, significantly decreased mean cell haemoglobin accompanying the elevated numbers of erythrocytes in the combination-treated mice was found. This effect could be interpreted as the result of a sublethal 5-FU-induced damage to erythroid progenitor and precursor cells forced to proliferate intensively by the combination therapy. The observed additivity and synergism of G-CSF with elevated extracellular adenosine in terms of erythropoiesis is an interesting finding with potential implications in clinical practice.

Activation of metabotropic glutamate receptors increases extracellular adenosine in vivo.

In order to identify the mechanisms that would lead to increased levels of the inhibitory neuromodulator adenosine in the brain, we tested metabotropic glutamate receptor agonists for their ability to increase extracellular adenosine in the cortex of unanesthetized rat using in vivo microdialysis. The group I/II metabotropic glutamate receptor agonist trans-(+/-)- 1-amino-(1S,3R)-cyclopenyanedicarboxylic acid (I mM) increased extracellular adenosine as did the specific group I agonist (S)- 3,5-dihydroxyphenylglycine (DHPG; 1 mM). The evoked increase of adenosine by 1 mM DHPG was reduced by the group I antagonist (RS)- 1-aminoindan-1,5,-dicarboxylic acid. Activation of group II or III metabotropic receptors did not affect extracellular adenosine. These results suggest that activation of group I metabotropic receptors contributes to elevated extracellular adenosine levels in vivo.

CFTR deltaF508 carrier status, risk of breast cancer before the age of 40 and histological grading in a population-based case-control study.

There has been recent interest in the risk of various cancers in cystic fibrosis (CF) patients and carriers of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. It has been proposed that a CFTR mutation may protect against breast cancer, based on evidence that elevated extracellular adenosine triphosphate (ATP) is known to inhibit breast cancer cell line growth and that CFTR pumps ATP out of epithelial cells. A CFTR mutation would therefore result in higher concentrations of serum ATP. A CFTR knockout mouse model had high serum concentrations of ATP and showed reduced breast tumour implantibility and decreased breast cancer growth rates. We have evaluated the relationship between the deltaF508 CFTR mutation and the risk of breast cancer before the age of 40. The deltaF508 CFTR mutation carrier rate in 272 cases (2.2%) was no different from the carrier rate observed in 171 controls (1.8%). If there was a protective effect resulting from the postulated elevation in serum ATP levels, tumours arising in deltaF508 CFTR carriers would have been expected to be generally less aggressive. When the histological features of the breast cancers with a deltaF508 CFTR mutation were reviewed and graded using a combined architectural and cytological grading system, all were found to be grade III, poorly differentiated tumours, contrary to the predictions. A combination of our data with other large population-based samples of cases and controls is required to resolve this issue.

Granulocyte colony-stimulating factor and drugs elevating extracellular adenosine synergize to enhance haematopoietic reconstitution in irradiated mice.

The activation of adenosine receptors has recently been demonstrated to stimulate haematopoiesis. In the present study, we investigated the ability of drugs elevating extracellular adenosine to influence curative effects of granulocyte colony-stimulating factor (G-CSF) in mice exposed to a sublethal dose of 4 Gy of 60Co radiation. Elevation of extracellular adenosine in mice was induced by the combined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole plus AMP, and G-CSF, administered either alone or in combination, were evaluated. The drugs were injected to mice in a 4-d treatment regimen starting on d 3 after irradiation and the haematopoietic response was evaluated on d 7, 10, 14, 18 and 24 after irradiation. While the effects of G-CSF on the late maturation stages of blood cells, appearing shortly after the completion of the treatment, were not influenced by dipyridamole plus AMP, positive effects of the combination therapy occurred in the post-irradiation recovery phase which is dependent on the repopulation of haematopoietic stem cells. This was indicated by the significant elevation of counts of granulocyte-macrophage progenitor cells (GM-CFC) and granulocytic cells in the bone marrow (d 14), of GM-CFC (d 14), granulocytic and erythroid cells (d 14 and 18) in the spleen, and of neutrophils (d 18), monocytes (d 14 and 18) and platelets (d 18) in the peripheral blood. These effects suggest that the repopulation potential of the combination therapy lies in a common multilineage cell population. The results of this study implicate the promising possibility to enhance the curative effects of G-CSF under conditions of myelosuppressive states induced by radiation exposure.

Determination of adenosine deaminase binding domain on CD26 and its immunoregulatory effect on T cell activation.

CD26, a 110-kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV enzyme activity and plays an important role in T cell costimulation. In the present study, we examined both the exact adenosine deaminase (ADA) binding domain on CD26 and the functional consequences of mutated CD26 transfectants that were deficient for cell surface ADA. Using CD26 deletion, human-rat swap, and point mutations, we found that the residues of L340, V341, A342, and R343 on the CD26 molecule were essential amino acids for ADA binding. When these amino acids were mutated and transfected into Jurkat cells, the resultant CD26 transfectants expressed only CD26, not ADA, on the cell surface. The amount of IL-2 produced by wild-type and mutated CD26 transfectants was almost the same following stimulation with anti-CD3 plus PMA. However, the mutated CD26 transfectants were much more sensitive to the inhibitory effect of adenosine on IL-2 production than were the wild CD26 transfectants. These data suggest that ADA on the cell surface does not directly involve T cell activation. Conversely, CD26 alone does not result in modulating the inhibitory effect of adenosine. Only the ADA bound to CD26 on the cell surface was functional and could counteract the inhibitory effect of elevated extracellular adenosine.

Support of homeostatic glial cell signaling: a novel therapeutic approach by propentofylline.

A pathological glial cell activation, which forces microglia to transform into immunocompetent cells with cytotoxic properties and astrocytes to "de-differentiate," presumably adds to neurodegenerative diseases. We examined the modulatory effect of adenosine on the Ca2+ and cAMP-dependent regulation of such reactive glial cell properties in culture and tested possibilities of pharmacologic reinforcement. A strengthening of the cAMP-signaling, as could be achieved by adenosine agonists via a Ca(2+)-dependent action, favored the differentiation of proliferating astrocytes and associated neuroprotective properties (ion homeostasis, formation of trophic factors). But potentially neurotoxic properties of microglial cells were inhibited. Adenosine depressed their proliferation rate and transformation into macrophages, their particularly high formation of reactive oxygen intermediates and the release of the cytokine TNF-alpha. Similar effects were obtained with propentofylline, which acts as selective cAMP/cGMP phosphodiesterase inhibitor and also increases the effective concentration of adenosine by blocking its cellular reuptake. The recently observed induction of microglial apoptosis by elevated extracellular adenosine levels may further contribute to limit secondary nerve cell damage related to a pathological glial cell activation.

Synergistic Effect of Granulocyte Colony-Stimulating Factor and Drugs Elevating Extracellular Adenosine on Neutrophil Production in Mice

Experimental evidence suggests that the activation of purinoceptors by extracellular adenosine can modulate proliferation and/or differentiation of hematopoietic cells. The present study was undertaken to investigate the potential interactions of this system of intercellular signaling with the effects of granulocyte colony-stimulating factor (G-CSF) on granulopoiesis in vivo. Elevation of extracellular adenosine in normal mice was induced by the joined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole, AMP, and G-CSF, administered either alone or in combinations, were evaluated. The agents were injected to mice in a 4-day regimen, and the hematologic endpoints were determined 24 hours after the completion of the treatment. It was shown that the effects of G-CSF, ie, increases in peripheral blood neutrophils, granulocyte-macrophage progenitor cells (GM-CFC), and morphologically determined granulocytic cells in femoral marrow and a decrease in the marrow erythroid cells, can be enhanced by the combination of dipyridamole plus AMP administered 30 minutes before G-CSF. Furthermore, it was ascertained that the stimulatory action of dipyridamole plus AMP was expressed particularly at lower doses of G-CSF (1.5, 3, and 4.5 micrograms/d). At higher doses of G-CSF (6 and 9 micrograms/d), the interactions were no more evident. When combining dipyridamole, AMP, and 3 micrograms of G-CSF, peripheral neutrophils increased approximately 3.9- to 4.5-fold compared with an approximate 2.2-fold increase induced by G-CSF alone. The results indicate the possible therapeutic potential of combination therapy with G-CSF and drugs increasing extracellular adenosine.