Enzymes of adenosine metabolism in lymphocytes and the functional state of the sympatho-adrenal system in tumor processes

A correlation between reactions of the sympathoadrenal system and the activity of adenosine transformation enzymes in lymphocytes is demonstrated in the dynamics of metastatic Lewis carcinoma development in C57Bl mice. In the period when metastases arise a decrease in the adenosine deaminase activity in lymphoid cells of the thymus and spleen is accompanied by drop in the content of DOPA, noradrenalin and adrenalin in adrenals. At the late stages of the tumour process a decrease in the amount of these compounds in adrenals is accompanied by the diminution of the adenosine deaminase activity and by an increase in the 5'-nucleotidase activity in the thymus. Contrary changes are observed in spleen lymphocytes. The revealed disturbances may stimulate to a considerable extent the appearance and growth of metastases.     

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Effect of splenin and splenic protein-free extracts on the adenosine deaminase activity in various organs of the rat

It has been shown that the adenosine deaminase activity of intact rats increases in such lymphoid organs as the thymus and spleen under the influence of splenic protein-free extracts dissolved in the ratio of 1:100. The enzyme activity in testes does not change but its decrease (by 26.2%) is observed in the adrenal glands under the influence of the splenic protein-free extract. An analogous effect is revealed in splenin and its fractions. The splenic protein-free extract increases (by 83%) the enzyme activity in the thymus of splenectomized rats as compared to intact animals but does not change it in a homogenate of testes.     

Immunomorphological localization of adenosine deaminase in rat tissues during ontogeny

Summary Immunomorphological methods were used to localize adenosine deaminase in tissues of the rat at different stages of ontogeny. In the thymus, lymphocytes began to express significant amounts of the enzyme with the appearance of demarcation between the cortex and medulla at 17 days of gestation. At any stage of ontogeny studied, strong adenosine deaminase staining was seen predominantly in cortical thymocytes. In the spleen and lymph node, the enzyme was initially detected in T cell areas, whereas primary follicles did not show positive adenosine deaminase staining. During further development, the enzyme was demonstrated in some lymphocytes of germinal centres and plasma cells. In the duodenum, epithelial cells of villi and the neck of crypts showed positive adenosine deaminase staining whereas no staining for the enzyme was observed in the epithelial cells of the base of crypts. Strongly positive staining for adenosine deaminase appeared in plasma cells of the lamina propria by four weeks after birth. The transient positive reaction for the deaminase could be recognized in epithelial cells of tubules of the kidney during late foetal and early postnatal development. The tubules of adult rats did not stain for the enzyme. In the cartilage of 15-day foetuses, positive adenosine deaminase staining was seen only in perichondrial cells and hypertrophic cells. Kuppfer cells in the liver and endothelial cells of blood vessels stained positively for the enzyme at every stage of ontogeny studied.     

The pool of free purine and pyrimidine nucleosides and bases in the thymocytes, and splenic T- and B-lymphocytes of C3HA mice during the growth of solid hepatoma 22a

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.     

Lack of adenosine deaminase deficiency in the mutant mouse wasted

The possibility that the mutant mouse wasted (wst/wst) may serve as an animal model for studies of severe combined immunodeficiency disease (SCID) and the role of adenosine deaminase (ADA, EC 3.5.4.4) in adenosine metabolism were investigated. The specific activity of ADA in wst/wst compared with control mice was significantly lower by 26% in thymus, but significantly higher by 18% in spleen and 32% in cerebellum. Vmax values of ADA in spleens were 43% higher in wst/wst mice and no changes were observed in Km values. In contrast, the Vmax of ADA was unchanged in erythrocytes from wst/wst mice, but the Km for adenosine was significantly elevated. Thus, based on ADA measurements alone, it may be premature to consider wst/wst mice as a model for ADA deficiency and SCID in humans.     

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.     

Xanthine oxidase and adenosine deaminase in commercial bovine spleen phosphodiesterase preparations.

Commercial bovine spleen phosphodiesterase preparations contain xanthine oxidase activity; the xanthine oxidase in such preparations mediates the oxidation of a pteridine derivative as well as a standard purine substrate (hypoxanthine). The xanthine oxidase activity in the phosphodiesterase preparations is inhibited strongly by allopurinol (4-hydroxypyrazolo(3,4-d) pyrimidine). The reported ability of phosphodiesterase preparations to catalyze the deamination of adenosine derivatives appears to be due to contamination with a conventional adenosine deaminase in view of the observations that this activity is inhibited by an established inhibitor of adenosine deaminase and that the relative rates of deamination of N¹-methyladenosine and adenosine are similar with both the phosphodiesterase preparation and calf intestine adenosine deaminase.     

Expression of terminal deoxynucleotidyl transferase in human thymus during ontogeny and development

Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in human thymus during ontogeny and development. In five fetal thymus samples, the enzyme activity was barely detectable. At birth, the terminal transferase activity remained low. Maximum expression of the enzyme activity occurred between 10 and 40 mo of age. Analysis of six other enzyme activities, adenosine kinase, deoxyadenosine kinase, AMP deaminase, dAMP deaminase, 5' nucleotidase, and adenosine deaminase confirmed the normal status of the thymic tissue. A careful analysis of thymic architecture revealed that involution did not occur as a result of the disease process that necessitated cardiac surgery. By immunofluorescence, the TdT antigen was localized exclusively in the nucleus of cortical thymocytes. Protein immunoblotting studies indicated that human thymic terminal transferase exists as a single high m.w. species in individuals under 30 mo of age. Thereafter, a variant m.w. species is detectable. The increase in expression of this enzyme coincides with the increase observed in serum immunoglobulin levels during maturation and precedes the maximum development of the human thymus.     

Adenosine signaling and adenosine deaminase regulation of immune responses: impact on the immunopathogenesis of HIV infection

Infection by human immunodeficiency virus (HIV) causes the acquired immune deficiency syndrome (AIDS), which has devastating effects on the host immune system. HIV entry into host cells and subsequent viral replication induce a proinflammatory response, hyperactivating immune cells and leading them to death, disfunction, and exhaustion. Adenosine is an immunomodulatory molecule that suppresses immune cell function to protect tissue integrity. The anti-inflammatory properties of adenosine modulate the chronic inflammation and immune activation caused by HIV. Lack of adenosine contributes to pathogenic events in HIV infection. However, immunosuppression by adenosine has its shortcomings, such as impairing the immune response, hindering the elimination of the virus and control of viral replication. By attempting to control inflammation, adenosine feeds a pathogenic cycle affecting immune cells. Deamination of adenosine by ADA (adenosine deaminase) counteracts the negative effects of adenosine in immune cells, boosting the immune response. This review comprises the connection between adenosinergic system and HIV immunopathogenesis, exploring defects in immune cell function and the role of ADA in protecting these cells against damage.     

Inverse relationship between adenosine deaminase and purine nucleoside phosphorylase in rat lymphocyte populations

The levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in rat lymphoid cell populations. The specific activities of the two enzymes in thymus, lymph node, spleen, and bone marrow were found in inverse proportion to each other. Thy mocytes had the highest ADA activity and the lowest PNP activity, whereas spleen and bone marrow lymphocytes had the lowest ADA activity (six- to sevenfold lower than thymocytes) and the highest PNP activity (fourfold higher than thymocytes). This reciprocal relationship was also found in cells of the T lymphocyte lineage at various stages of differentiation. These results suggest that specific stages of T-cell development may be characterized by the levels of the two enzymes, and that deficiencies of each of these enzymes might affect T cells at separate stages of differentiation.     

Inhibition of adenosine deaminase leads to enhanced antibody responses in the mouse

Inhibition of adenosine deaminase activity leads to decreased cellular immunity. The effect of deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase, on the ability of mouse spleen cells to generate antibody responses in vitro has been examined. With either continuous exposure to or pretreatment of the cells with deoxycoformycin, there was a decrease in cell survival and an increase in antibody-producing cells in the surviving cell population. To identify the cell population most susceptible to the inhibitor, the spleen was separated into B-cell, and T-cell, and macrophage components and each population was pretreated with deoxycoformycin before combination with its complementary treated or untreated population. Deoxycoformycin pretreatment had no effect on macrophages or B cells; however, pretreatment of the T cells resulted in increased antibody responses. When T cells and B cells were both pretreated and combined, there was a synergistic increase in the antibody response. In addition, supernatants from cultures in which both B cells and T cells had been pretreated with DCF were capable of enhancing antibody responses in cultures containing DCF-treated T cells. Though adenosine was increased in the stimulatory culture supernatants, adenosine alone did not enhance antibody responses in either untreated or DCF-treated cultures.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Immunoassay of the adenosine deaminase complexing proteins of human tissues and body fluids.

A sensitive immunoassay for the adenosine deaminase binding protein (complexing protein) of human kidney has been developed. Impetus for the development of the assay was provided by the observations that (a) antibody to complexing protein does not react with the catalytically active adenosine deaminase monomer, and (b) binding of antibody to complexing protein does not affect the binding or catalytic activity of the enzyme monomer. Preformed immune precipitate prepared from rabbit anti-kidney complexing protein serum and goat anti-rabbit gamma-globulin serum is used to selectively insolubilize complexing protein. Quantitation is accomplished by measuring the intrinsic adenosine deaminating activity or adenosine deaminase binding capacity of the protein held in the immune precipitate. As little as 1 ng of kidney complexing protein can be accurately quantitated with the assay. The assay was used to demonstrate that complexing proteins from liver, lung, spleen, fibroblasts, plasma, and urine react with antibody to kidney complexing protein. The shared capacity to bind adenosine deaminase coupled with their antigenic similarity suggests that the complexing proteins of a number of human tissues and body fluids may be products of the same gene.     

Purine metabolizing enzyme activities in radiosensitive tissues of mice after sublethal whole-body irradiation

The activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were determined between days 1-14 in the spleen, thymus and femoral bone marrow of mice subjected to whole-body gama irradiation with a dose of 5.5 Gy. In control animals, the highest activity of ADA (as related to 10(6) cells) was recorded in the thymus (58.9 pmol.s-1), the lowest one in the femur (34.8 pmol.s-1), the PNP activity was the lowest in the thymus (14.5 pmol.s-1) and the highest in the femur (96.0 pmol.s-1). In the spleen, an elevation of ADA activity (up to 379%) was observed during the first postirradiation days; PNP activity was reduced (to 58%) on postirradiation day 3, followed by the return and even elevation on day 14 (265%). In the thymus, a parallel reduction of the activities of both enzymes appeared during the first postirradiation days, with a subsequent increase during the regeneration phase. In the femoral bone marrow, ADA and PNP activities were increased on postirradiation day 1 (275% and 201%, respectively). Reference is made to the possible relationship between the observed characteristic changes in activities and the degree of damage and/or renewal of cell population in the hemopoietic tissues after irradiation.     

Effect of splenectomy on the development of neonatal lymphoid organs transplanted to animals of varying age]

The effect of splenectomy on the development of newborn thymus and spleen grafted under the kidney capsule of young and old mice has been investigated. Preliminary splenectomy is shown to increase cell counts in grafted spleen that is more conspicuous in young recipients as compared with old ones. This result suggests a decrease with age in the inhibitory effect of the host spleen on the maturation of spleen grafted from newborn donor. Combined transplantation of newborn thymus and spleen has revealed a decrease of cell counts in the donor spleen grafted to the young splenectomized recipients and, on the contrary, increase of this parameter in old ones. Immune response in donor spleen with combined transplantation of the thymus to the old splenectomized recipients is much higher as compared with the same parameter in recipient without splenectomy. It is concluded that partial destruction of the old immune system is essential for its correction.     

Effects of the age of the recipient on the outcome of transplantation of lymphoid organs from newborn donors

The comparison of developmental capacities of simultaneously grafted thymus and spleen taken from newborn CBA mice in young and old macroenvironment was attempted using the method of heterotopic chimera construction. The perceptible augmentation of immune response in both host and donor spleens was obtained in case of transplantation into the young animals, and completely opposite effect, i. e. well-marked immunosuppression in case of old recipients. Preliminary removal of host spleen results in abolition of this suppressive effect in old animals. Moreover, immune response in donor spleen following thymus transplantation into the old splenectomized recipients was significantly higher compared to the same one without additional thymus graft. Taken together, these findings indicate that despite the existence of some potency for autonomous development the eventual effect of neonatal lymphoid organs maturation strongly depends upon the age of system in which this maturation does take place.     

The role of the thymus in the establishment of suppressor cells in liver chimeras.

The role of the thymus in the establishment of specific suppression was studied in an experimental model in which lethally irradiated F1 mice were reconstituted with parental C57BL neonatal liver cells and then challenged with immunocompetent spleen cells syngeneic to the donor. In this model, inhibition of a graft-versus-host response by these spleen cells served as an indication of the establishment of a suppressive state towards syngeneic antigens in the liver chimeras. It was demonstrated that since thymectomy of the chimeras could not prevent the elicitation of a graft-versus-host response by spleen cells, the thymus is essential for the establishment of this specific suppression. Reconstitution of such chimeras with intact thymus grafts of either donor (C57BL) or host (F1) origin led to the inhibition of the graft-versus-host response and to the reappearance of the suppressive state. Removal of the thymus in intact liver chimeras after establishment of a suppressive state did not affect suppression. Thus, it was concluded that the thymus is needed in the chimeras during a critical period in the development of suppression. Once suppression is established, the presence of the thymus is no longer required.     

Immunoaffinity purification and fluorescence studies of human adenosine deaminase

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.