Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation, and airway obstruction.

Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologically active purines adenosine and 2'-deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient mice included an accumulation of activated alveolar macrophages and eosinophils. These changes were accompanied by a pronounced enlargement of alveolar spaces and increases in mucus production in the bronchial airways. The alveolar enlargement was found to be due in part to abnormal alveogenesis. Lowering adenosine and 2'-deoxyadenosine levels using ADA enzyme therapy decreased the pulmonary eosinophilia and resolved many of the lung histopathologies. In addition, genetically restoring ADA to the forestomach of otherwise ADA-deficient mice prevented adenine metabolic disturbances as well as lung inflammation and damage. These data suggest that disturbances in purinergic signaling mediate the lung inflammation and damage seen in ADA-deficient mice.

Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling.

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase-deficient fetal thymic organ cultures.

Murine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2'-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vbeta gene rearrangements and pre-TCR-alpha expression were normal in ADA-deficient cultures, the production of alphabeta TCR(+) thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of beta selection. In contrast, the production of gammadelta TCR(+) thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor-1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as beta selection.

Murine ecto-5'-nucleotidase (CD73): cDNA cloning and tissue distribution.

The murine cDNA, encoding the purine catabolic enzyme, ecto-5'-nucleotidase (NT), was cloned and the tissue-specific distribution of both the mRNA and enzyme activity was examined. Starting with kidney RNA and primers based on the known rat sequence, reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to obtain the complete sequence for the translated portion of the murine cDNA. Murine NT is 94% identical to human NT at the amino acid (aa) level and 86% identical at the nucleotide (nt) level. NT enzyme assays revealed greater than tenfold more NT activity in mature vs. immature murine T- and B-lymphocytes. A similar increase in NT activity was also found when the pre-B-cell line, 70Z/3, was induced to produce surface kappa light chains with lipopolysaccharide (LPS) and gamma-interferon (gamma-IFN). Thus, culture systems in which murine lymphocytes mature may be useful for examining the mechanisms of NT gene regulation, as well as the function of NT in the immune system. In tissues, enzyme activity varied over 30-fold, from the lowest levels in skeletal muscle, thymus and spleen to highest in placenta, kidney and forestomach. Levels of mRNA, as determined by RNase protection assay, showed increased NT expression in the early gestation site, as compared to non-pregnant uterus, and in day-19.5 placenta, as compared to day-13 chorioallantoic placenta. Messenger RNA levels were in general proportional to enzyme activity, except in the lung and glandular stomach where mRNA levels were higher than expected, based on enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A1 adenosine receptors mediate hypoglycemia-induced neuronal injury.

The cellular mechanisms that lead to neuronal death following glucose deprivation are not known, although it is recognized that hypoglycemia can lead to perturbations in intracellular calcium ([Ca2+]i) levels. Recently, activation of A1 adenosine receptors (A1AR) has been shown to alter [Ca2+]i and promote neuronal death. Thus, we examined if A1AR activation contributes to hypoglycemia-induced neuronal injury using rat cortical neurons. First, we observed that hypoglycemia was associated with large increases in neuronal adenosine release. Next, decreased neuronal viability was seen with progressive reduction in glucose concentration (25, 6, 3, 0.75 and 0 mM). Using the calcium-sensitive dye, Fluo-3, we observed both acute and long-term changes in relative [Ca2+]i during hypoglycemic conditions. Demonstrating a role for adenosine in this process, both the loss in neuronal viability and the early changes in [Ca2+]i were reversed by treatment with A1AR antagonists (8-cyclopentyl, 1,3-dipropylxanthine; 9-chloro-2-(2-furyl)(1,2,4)-triazolo(1,5-c)quinazolin-5-amine; and N-cyclopentyl-9-methyladenine). We also found that hypoglycemia induced the expression of the pro-apoptotic enzyme, caspase-3, and that A1AR antagonism reversed hypoglycemia-induced caspase-3 activity. Collectively, these data show that hypoglycemia induces A1ARs activation leading to alterations in [Ca2+]i, which plays a prominent role in leading to hypoglycemia-induced neuronal death.

Adenosine deaminase-deficient mice generated using a two-stage genetic engineering strategy exhibit a combined immunodeficiency.

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency. The mechanisms involved in the lymphoid specificity of the disease are not fully understood due to the inaccessibility of human tissues for detailed analysis and the absence of an adequate animal model for the disease. We report the use of a two-stage genetic engineering strategy to generate ADA-deficient mice that retain many features associated with ADA deficiency in humans, including a combined immunodeficiency. Severe T and B cell lymphopenia was accompanied by a pronounced accumulation of 2'-deoxyadenosine and dATP in the thymus and spleen, and a marked inhibition of S-adenosylhomocysteine hydrolase in these organs. Accumulation of adenosine was widespread among all tissues examined. ADA-deficient mice also exhibited severe pulmonary insufficiency, bone abnormalities, and kidney pathogenesis. These mice have provided in vivo information into the metabolic basis for the immune phenotype associated with ADA deficiency.

Activation of apoptosis in early mouse embryos by 2'-deoxyadenosine exposure.

Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamination of adenosine and deoxyadenosine to nontoxic derivatives. The importance of this reaction in the female reproductive tract of mice is suggested by pronounced utero-placental expression of ADA, and by embryolethality of the potent ADA-inhibitor deoxycoformycin (dCF) on day 7-8 of gestation. The present study investigated the effects of dCF, adenosine, and deoxyadenosine on the mouse neurula. Morphological cell death was monitored by the acridine orange reaction (AOR), and biochemical cell death by internucleosomal DNA cleavage (IDC). A strong AOR appeared in day 7-8 embryos between 3 and 4.5 hr post-exposure to dCF in utero; there was no apparent effect on day 6 or day 9 embryos. Most embryonic tissues were responsive, although the heart and extraembryonic membranes were resistant. Up to 75% of the embryonic chromatin was degraded in a regular pattern in concert with the AOR. Immediate activation of "whole-body" apoptosis was reproduced in short-term whole embryo culture with 0.1 mM deoxyadenosine in the presence of 0.01 mM dCF. This was not activated by exposure to dCF alone nor to adenosine; however, high concentrations of adenosine completely blocked the response to deoxyadenosine, whereas niacinamide inhibited the AOR without changing IDC. The cytotoxic effect of deoxyadenosine was correlated with an expansion of embryonic dATP pools determined by high-performance liquid chromatography analysis. The results suggest that deoxyadenosine is the embryotoxic metabolite which accumulates in the antimesometrium of pregnant mice treated with dCF. Exposure to this metabolic toxin activates apoptosis in day 7-8 embryos through an adenosine-sensitive, NAD-dependent mechanism.

Genetically engineered mice demonstrate that adenosine deaminase is essential for early postimplantation development.

Adenosine deaminase (ADA) is an essential enzyme of purine metabolism that is enriched at the maternal-fetal interface of mice throughout postimplantation development. During early postimplantation stages Ada is highly expressed in both maternally derived decidual cells and zygotically derived trophoblast cells. For the current study we utilized genetically modified mice to delineate the relative contribution and importance of decidual and trophoblast ADA at the maternal-fetal interface. In females genetically engineered to lack decidual ADA a striking pattern of expression was revealed in giant trophoblast cells that surround the early postimplantation embryo. Embryos within gestation sites lacking both decidual and trophoblast ADA died during the early postimplantation period, whereas expression in trophoblast cells alone was sufficient for survival through this period. Severe disturbances in purine metabolism were observed in gestation sites lacking decidual ADA, including the accumulation of the potentially toxic ADA substrates adenosine and 2'-deoxyadenosine. These experiments provide genetic evidence that Ada expression at the maternal-fetal interface is essential for early postimplantation development in mice.

The importance of adenosine deaminase for lymphocyte development and function.

Deficiency in the enzyme adenosine deaminase (ADA) in humans manifests primarily as severe lymphopenia and immunodeficiency, resulting in death by 6 months of age, if untreated. In this review, we discuss phenotypical, biochemical, and metabolic hallmarks of the disease, and describe a mouse model in which levels of ADA can be biochemically and genetically manipulated. This model provides exciting possibilities for uncovering the mechanisms by which this purine catabolic enzyme affects lymphopoiesis.

Apneas and oscillation of cardiac ectopy in Cheyne-Stokes breathing during sleep.

Previous investigators have described heart rate oscillations (1,2) and various arrhythmias (3-5) during Cheyne-Stokes breathing. To our knowledge, however, this is the first report describing cardiac ectopic beats oscillating with the identical frequency as ventilatory movement and SaO2 in Cheyne-Stokes breathing during sleep. This case is also the first to document with polygraphic sleep recordings the suggested efficacy of acetazolamide (26) in abolishing the apneas of Cheyne-Stokes breathing during sleep. However, generalizations regarding the impact of ventilation on cardiac ectopy and the therapeutic effect of acetazolamide will require prospective studies on multiple patients with Cheyne-Stokes breathing during sleep.

Metabolic and immunologic consequences of limited adenosine deaminase expression in mice.

Adenosine deaminase (ADA; EC 3.5.4.4) deficiency in humans is an autosomal recessive genetic disorder that results in severe combined immunodeficiency disease. ADA-deficient mice generated by targeted gene disruption die perinatally, preventing postnatal analysis of ADA deficiency. We have recently rescued ADA-deficient fetuses from perinatal lethality by expression of an ADA minigene in the placentas of ADA-deficient fetuses, thus generating postnatal mice admissible to analysis of ADA deficiency. The minigene used also directed ADA expression to the forestomach postnatally, producing adult animals that lacked ADA enzymatic activity in all tissues outside the gastrointestinal tract. Mice with limited ADA expression exhibited profound disturbances in purine metabolism, including thymus-specific accumulations of deoxyadenosine and dATP, and inhibition of S-adenosylhomocysteine hydrolase in the thymus, spleen, and, to a lesser extent, the liver. Lymphopenia and mild immunodeficiency were associated with these tissue-specific metabolic disturbances. These mice represent the first genetic animal model for ADA deficiency and provide insight into the tissue-specific requirements of ADA.

Tissue-specific rescue suggests that placental adenosine deaminase is important for fetal development in mice.

Adenosine deaminase (ADA, EC 3.5.4.4) is an essential enzyme of purine metabolism that is expressed at very high levels in the murine placenta where it accounts for over 95% of the ADA present at the fetal gestation site. We have recently shown that ADA-deficient fetuses, which also lack ADA in their adjoining placentas, die during late fetal development in association with profound purine metabolic disturbances and hepatocellular impairment. We have now investigated the potential importance of placental ADA by genetically restoring the enzyme to placentas of ADA-deficient fetuses. This genetic engineering strategy corrected most of the purine metabolic disturbances, prevented serious fetal liver damage, and rescued the fetuses from perinatal lethality. Our findings suggest that placental ADA is important for murine fetal development and illustrate a general strategy for the tissue specific correction of phenotypes associated with null mutations in mice.

Adenosine-mediated mast cell degranulation in adenosine deaminase-deficient mice.

Adenosine is a signaling nucleoside that has been suggested to play a role in asthma in part through its ability to influence mediator release from mast cells. Adenosine levels are elevated in the lungs of asthmatics, further implicating this molecule in the regulation of lung inflammation and suggesting that animal models exhibiting endogenous increases in adenosine will be useful for the analysis of adenosine function. Adenosine deaminase (ADA) is a purine catabolic enzyme responsible for regulating the levels of adenosine in tissues and cells. ADA-deficient mice develop lung inflammation and damage reminiscent of that seen in asthma in association with elevated adenosine levels. In the current study, we investigated the status of mast cells in ADA-deficient lungs. ADA-deficient mice exhibited extensive lung mast cell degranulation concurrent with elevated adenosine levels. ADA enzyme therapy prevented the accumulation of lung adenosine as well as mast cell degranulation, suggesting that this process was dependent on elevated lung adenosine levels. Consistent with this, treatment of ADA-deficient mice with broad spectrum adenosine receptor antagonists attenuated degranulation by 30 to 40%, supporting the involvement of adenosine receptor signaling. Moreover, these studies demonstrate the ability of endogenously generated adenosine to influence lung mast cell degranulation in a receptor-mediated manner and establish ADA-deficient mice as a model system to investigate the specific adenosine receptor responses involved in the degranulation of lung mast cells.

Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase.

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Diverse genetic regulatory motifs required for murine adenosine deaminase gene expression in the placenta.

Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.

Adenosine-dependent airway inflammation and hyperresponsiveness in partially adenosine deaminase-deficient mice.

Adenosine is a signaling nucleoside that is elevated in the lungs of asthmatics. We have engineered a mouse model that has elevated levels of adenosine as a result of the partial expression of the enzyme that metabolizes adenosine, adenosine deaminase (ADA). Mice with lowered levels of ADA enzymatic activity were generated by the ectopic expression of an ADA minigene in the gastrointestinal tract of otherwise ADA-deficient mice. These mice developed progressive lung inflammation and damage and died at 4-5 mo of age from respiratory distress. Associated with this phenotype was a progressive increase in lung adenosine levels. Examination of airway physiology at 6 wk of age revealed alterations in airway hyperresponsiveness. This was reversed following the lowering of adenosine levels using ADA enzyme therapy and also through the use of the adenosine receptor antagonist theophylline, implicating both the nucleoside and its receptors in airway physiological alterations. All four adenosine receptors were expressed in the lungs of both control and partially ADA-deficient mice. However, transcript levels for the A(1), A(2B), and A(3) adenosine receptors were significantly elevated in partially ADA-deficient lungs. There was a significant increase in alveolar macrophages, and monocyte chemoattractant protein-3 was found to be elevated in the bronchial epithelium of these mice, which may have important implications in the regulation of pulmonary inflammation and airway hyperresponsiveness. Collectively, these findings suggest that elevations in adenosine can directly impact lung inflammation and physiology.