IFN-beta regulates CD73 and adenosine expression at the blood-brain barrier

IFN-beta treatment reduces the relapse rate in MS but its mechanism of action remains incompletely understood. Our aim was to clarify the beneficial effect of IFN-beta in the treatment of MS. We assessed the influence of IFN-beta treatment on (i) CD73 expression on the surface of primary cultures of human blood-brain barrier endothelial cells (BBB-EC) and human astrocytes using immunofluorescence staining and flow cytometry, (ii) transmigration of CD4+ T lymphocytes using an in vitro model of BBB and (iii) CD73 enzyme activity, i.e. ecto-5'-nucleotidase activity in the serum of MS patients using a radiochemical assay. IFN-beta increases the expression of ecto-5'-nucleotidase both on BBB-EC and astrocytes. As a consequence, lymphocyte transmigration through BBB-EC is reduced. Importantly, this reduction can be reversed using alpha,beta-methyleneadenosine-5'-diphosphate, a specific inhibitor of ecto-5'-nucleotidase. CD73 is strongly expressed in microvasculature in samples of postmortem MS brain and, moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto-5'-nucleotidase activity and skin microvascular CD73 expression after IFN-beta treatment. Upregulation of ecto-5'-nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFN-beta on MS via enhancing the endothelial barrier function.     

Role of inositol starvation on ecto-5'-nucleotidase activity during mitogen-induced lymphocyte activation

Cell-surface 5'-nucleotidase was assayed by incubating intact cells with 5' [3H]AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell surface 5'-nucleotidase in small resting lymphocytes and in cells of the B cell line BCL1 was 5.7 and 1.1 nmol/min/mg protein respectively at 37 degrees C. The 5'-nucleotidase was inhibited by Concanavalin A and anti-IgM, the inhibition by anti-IgM being reversible. Incubating the lymphocytes in the presence and absence of mitogens in inositol-free medium for 15 min, 60 min, and 24 h had no effect on 5'-nucleotidase activity. The reaction product adenosine as well as adenine nucleotides were shown to inhibit mitogen-induced proliferation.     

Increased serum adenosine deaminase activity in schizophrenic receiving antipsychotic treatment

Adenosine is an important modulator of the nervous system that has been implicated in the pathophysiology of schizophrenia. We studied peripheral adenosine metabolism by determining the activity of serum adenosine deaminase, which converts adenosine into inosine, and 5'-nucleotidase, which converts AMP into adenosine, in 26 DSM-IV male schizophrenic patients under antipsychotic monotherapy and 26 healthy volunteers balanced for age and race. Schizophrenic patients treated either with typical antipsychotics or clozapine showed increased serum adenosine deaminase activity compared to controls (controls=18.96+/-4.61 U/l; typical=25.09+/-10.98 U/l; clozapine=30.32+/-10.83 U/l; p<0.05, ANOVA) and 5'-nucleotidase activity was also increased in patients on clozapine. After adjusting for confounding factors, adenosine deaminase, but not 5'-nucleotidase, alterations remained significant particularly in the clozapine group. This result suggests that either altered adenosine metabolism is present in schizophrenic patients or is influenced by treatment with antipsychotics, particularly clozapine.     

Age-related changes in adenosine metabolic enzymes in sleep/wake regulatory areas of the brain

The impact of age on the enzymatic activities of adenosine metabolic enzymes, i.e., adenosine deaminase, adenosine kinase, cytosolic- and ecto-5'-nucleotidase have been assessed in the brain sleep/wake regulatory areas of young, intermediate and old rats (2, 12 and 24 months, respectively). There were significant spatial differences in the distribution of enzymes of adenosine metabolism in the brain. Age did not impact on the enzymatic activity of adenosine deaminase. Adenosine kinase activity increased significantly in the cerebral cortex of old animals. However, there were no differences in the activity of adenosine kinase between young and intermediate aged rats. The largest age-related changes were in the activity of cytosolic- and ecto-5'-nucleotidase and there was a significant age-related increase in the activity of these enzymes in the sleep/wake regulatory areas. In addition, the activity of cytosolic- and ecto-5'-nucleotidase increased in the cerebral cortex of old and intermediate age rats when compared to young animals. An increase in the enzymatic activities in the cerebral cortex of adenosine kinase and 5'-nucleotideases was accompanied by an increase in the level of their mRNA. An increase in the activity of 5'-nucleotideases with age likely leads to an increase in adenosine levels in the brain.     

Anatomical and cytochemical relationships of adenosine deaminase-containing primary afferent neurons in the rat

The distribution of adenosine deaminase-containing neurons and fibers in the spinal cord and medulla was examined and the relationship of dorsal root ganglia neurons containing this enzyme to those containing somatostatin, substance P, fluoride-resistant acid phosphatase (FRAP) and 5'-nucleotidase was determined using immunohistochemical and histochemical methods. In the spinal cord adenosine deaminase-immunoreactive fibers and neurons were confined to layer I and IIo. A similar localization of these was observed in the spinal trigeminal nucleus. In adult animals treated neonatally with capsaicin adenosine deaminase-positive fibers were totally depleted in layer IIo but only partially depleted in layer I. Analysis of lumbar sensory ganglia revealed that small type-B neurons immunoreactive for adenosine deaminase were also immunoreactive for somatostatin but not substance P. In addition, adenosine deaminase-positive neurons lacked histochemical reaction-product for FRAP and exhibited the lowest activity of 5'-nucleotidase. Examination of the neuronal populations containing the two phosphatase enzymes showed that a proportion of neurons exhibiting 5'-nucleotidase activity were devoid of FRAP activity. It is concluded that dorsal root ganglia neurons immunoreactive for adenosine deaminase and somatostatin constitute a single subpopulation of type-B ganglion cells separate from those containing substance P or FRAP. It appears that the lack of coexistence of adenosine deaminase with either FRAP or 5'-nucleotidase cannot be attributed simply to a coexistence of the two latter enzymes since some 5'-nucleotidase-positive neurons lacking FRAP were also devoid of adenosine deaminase-immunoreactivity. Insofar as these three enzymes may contribute to the regulation of transmission processes in primary sensory neurons, our results indicate a minimal functional relationship between adenine nucleoside and nucleotide degrading enzymes in these neurons. In addition, FRAP appears to have some functional independence from 5'-nucleotidase.     

5'-Nucleotidase activity increases in aging rat brain.

The enzyme 5'-nucleotidase is present in glial and neuronal membranes, and catalyzes the formation of adenosine, which in turn can act as a neuromodulator or neurotransmitter. The present study found marked increases in histochemically demonstrated 5'-nucleotidase activity in most regions of rat brain from young adulthood (3-4 months) to middle age (12-18 months), with smaller or no changes between middle and old age (24-32 months). The aging adult cerebellum showed alterations in the histochemical pattern, with declines in the molecular layer and increases in the Purkinje layer. Both myelin and synaptic plasma membrane fractions from forebrain showed increases in enzyme activity. Assays of various other body tissues suggested that the increases are fairly specific to brain, and thus apparently do not represent a ubiquitous cellular mechanism of aging. Changes in brain 5'-nucleotidase activity during aging probably reflect the increasing number and size of glial cells, and perhaps also affect synaptic transmission through regulation of adenosine.     

Studies on the kinetic properties and subcellular localization of adenine nucleotide phosphatases in peripheral blood lymphocytes from control subjects and patients with common variable primary hypogammaglobulinaemia

Specific assays for 5'-nucleotidase, adenosine diphosphatase (ADPase) and Mg2+-dependent adenosine triphosphatase (Mg2+-ATPase) have been optimized for human lymphocytes and their subcellular localizations determined by sucrose density gradient centrifugation. 5'-Nucleotidase was localized solely to the plasma membrane of the lymphocyte. ADPase activity has been shown to have a dual localization to the plasma membrane and mitochondria, whilst Mg2+-ATPase was mainly located in the mitochondria. No [Na+,K+] activated Mg2+-dependent ATPase could be measured in these cells. We have confirmed the striking decrease in the specific activity of 5'-nucleotidase activity in lymphocytes from patients with common variable primary hypogammaglobulinaemia. In contrast, the specific activities of ADPase and Mg2+-ATPase showed no alteration in lymphocytes from the patient group when compared to controls. Thus the deficiency of ecto-5'-nucleotidase in the lymphocytes of patients with hypogammaglobulinaemia is a highly selective defect in purine metabolism.     

A quantitative histochemical study of 5'-nucleotidase activity in rat liver after ischaemia

The lead salt method of Wachstein and Meisel15 has been applied using incubation media containing polyvinyl alcohol for the localization and quantification of 5'-nucleotidase (E.C.3.1.3.5) activity in cryostat sections from rat liver after ischaemia in vitro and ischaemia in vivo followed by different periods of re-perfusion. 5'-Nucleotidase activity at the bile canaliculi, especially in the pericentral areas, had already decreased after 60 min of ischaemia in vitro, although the total activity as measured densitometrically was not changed. After 120-240 min of ischaemia, a significant decrease of the total 5'-nucleotidase activity was found. At that stage, signs of irreversible cell damage were recognized. Short periods of re-perfusion (1 h) after ischaemia in vivo induced a decreased bile canalicular 5'-nucleotidase activity throughout the entire liver, but a restoration after longer periods of re-perfusion was observed (5, 24, and 48 h). Necrotic areas recognized by a decreased lactate dehydrogenase activity after all periods of re-perfusion showed decreased total 5'-nucleotidase activities. A correlation was observed between the decrease in bile canalicular 5'-nucleotidase activity and the disappearance of microvilli of the bile canaliculi. It is concluded that a decrease in the bile canalicular 5'-nucleotidase activity can be used as a very sensitive marker for ischaemic liver cell damage. Assessment of the irreversibility of the cell injury has to be determined using additional parameters such as a decreased lactate dehydrogenase activity.     

Effect of chronic and acute stress on ectonucleotidase activities in spinal cord

We have previously observed that, while acute stress induces analgesia, chronic stress causes a hyperalgesic response in male rats. No effect was observed in females. There is increasing evidence that both ATP and adenosine can modulate pain. Extracellular ATP and ADP are hydrolyzed by an apyrase in synaptosomes from the peripheral and central nervous systems. In the present study, we investigated the effect of chronic and acute stress on ATPase-ADPase and 5'-nucleotidase activities in spinal cord of male and female rats. Adult male and female Wistar rats were submitted to 1 h restraint stress/day for 1 day (acute) or 40 days (chronic) and were sacrificed 24 h later. ATPase-ADPase activities were assayed in the synaptosomal fraction obtained from the spinal cord of control and stressed animals. ADP hydrolysis was decreased 25% in chronically stressed males, while no change was observed on ATPase activity. There was an increase in the 5'-nucleotidase activity in the same group. No effect on ADPase, ATPase or on 5'-nucleotidase activity was observed in females with chronic stress, or after acute stress neither in males or females. Chronic stress reduced ADP hydrolysis and increased 5'-nucleotidase activity in the spinal cord in male rats.     

Induction of ecto-5'-nucleotidase of rat cultured mesangial cells by interleukin-1 beta and tumour necrosis factor-alpha.

Because ecto-5'-nucleotidase activity of rat glomerular mesangial cells has been shown to increase upon interaction with macrophages in vitro, it was examined whether interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha), two macrophage-released cytokines, were responsible for this effect. IL-1 beta and TNF-alpha stimulated mesangial cell 5'-nucleotidase activity in a dose-dependent manner after treatment for 24 hr. Maximum increases reached 4.5 times and 1.7 times basal values for IL-1 beta (20 U/ml) and TNF-alpha (25 ng/ml), respectively. The effects of both cytokines were additive. Stimulation of 5'-nucleotidase by IL-1 beta and TNF-alpha was specific since the activity of other ectoenzymes, such as Mg2(+)-ATPase, was unchanged. Cycloheximide, a blocker of protein synthesis, suppressed the cytokine-dependent increase of 5'-nucleotidase activity. Cyclo-oxygenase inhibitors such as indomethacin and ibuprofen inhibited approximately 50% of the effects of both cytokines. Their inhibitory effect was abolished in the presence of prostaglandin E2 (PGE2). In addition, PGE2 itself produced a dose-related (0.1-10 microM) increase of 5'-nucleotidase activity with a maximum of 2.2 times basal value. Taken together, these results indicate that IL-1 beta, essentially, and TNF-alpha, to a lesser extent, regulate 5'-nucleotidase expression in the plasma membrane of cultured mesangial cells and that their effect depends in part on PGE2 synthesis. Therefore, macrophages, via their products of secretion acting on 5'-nucleotidase, could modulate adenosine production in the glomerular capillaries.     

5'-Nucleotidase activity of mossy fibers in the dentate gyrus of normal and epileptic rats.

Sprouting of mossy fibers in the hippocampus of rats that underwent limbic epileptogenesis by amygdala kindling or kainate injection was studied at the light microscopic and ultrastructural levels by cytochemical demonstration of the enzyme 5'-nucleotidase. This adenosine-producing ectoenzyme has previously been shown to characterize malleable terminals during brain development and lesion-induced synaptogenesis, but to be otherwise associated with glial membranes. At the light microscopic level, kainate-treated but not control or kindled rats showed 5'-nucleotidase activity in the CA3 region and in the inner molecular layer of the dentate gyrus. At the ultrastructural level, in control animals, the synapses of the molecular and granular layers were enzyme negative. Only some mossy fiber boutons of the dentate hilus exhibited 5'-nucleotidase activity. In epileptic rats, synaptic labeling within the hilus appeared more intense. Moreover, 5'-nucleotidase-containing terminals within the inner molecular layer, presumably ectopic mossy fiber boutons, were found in both kindled and kainate-treated rats. It is concluded that, in both the normal and epileptic hippocampus, 5'-nucleotidase is associated with axons capable of a plastic sprouting response. The synaptic enzyme may attenuate the glutamatergic transmission of mossy fibers, in particular of the aberrant mossy fibers in epileptic rats, by producing the inhibitory neuromodulator adenosine. Alternatively, 5'-nucleotidase may influence synapse formation by its putative non-enzymatic, adhesive functions.     

A B-cell hybridoma product inhibiting antibody secretion via 5''-nucleotidase.

A mouse spleen cell/plasmacytoma fusion designed to generate hybridomas making monoclonal antibodies to human lymphocyte 5'-nucleotidase yielded three hybridomas secreting material which inhibited about 50% of human and mouse lymphocyte ecto 5'-nucleotidase activity. The inhibition proved not to be due to antibody but to material of molecular weight 44,000 +/- 7,000 which was not part of an immunoglobulin molecule, although it may be related to a B cell Fc gamma receptor. In a haemolytic plaque assay this material inhibited the production of IgG but not IgM antibody by spleen cells of mice immunized with dinitrophenylated keyhole limpit haemocyanin. By contrast, IgG production by pokeweed mitogen-stimulated human tonsillar lymphocytes (assessed by reverse haemolytic plaque assay) was partially inhibited only by ascitic fluid of one of the hybridomas. The factor was called BAN (B cell anti 5'-nucleotidase). The suppressive action of BAN on IgG production was blocked by antibodies against 5'-nucleotidase suggesting that the lymphocyte enzyme may be acting as a BAN receptor.     

Ultrastructural localization of membrane phosphatases in teratocarcinoma and early embryos.

Ectodermal cells of the two- and three-germ layer-thick mouse egg-cylinders are considered to be the progenitors of embryonal carcinoma cells in embryo-derived teratocarcinomas. In an attempt to find differences between the tumor cells and equivalent embryonic cells, we have studied the electron microscopic cytochemical localization of alkaline phosphatase, 5'-nucleotidase, and Mg2+-activated adenosine triphosphatase (ATPase) in embryo-derived teratocarcinomas and mouse egg-cylinders. Alkaline phosphatase was detected in both embryonic and tumor cells, but its activity appeared much more intense in the tumor cells. No ATPase was demonstrated in embryonic ectodermal cells of 6-day-old embryos and only in occasional cells of 7- and 8-day-old embryos. No 5'-nucleotidase activity could be demonstrated in 6- to 8-day-old cylinders. There was marked ATPase and 5'-nucleotidase activity in the membranes of embryonal carcinoma cells. These data point out some differences on the plasma membrane between the embryonal carcinoma cells and equivalent embryonic cells. The potential significance of these differences is discussed with regards to the transformation of embryonic cells in tumor cells. (Am J Pathol 87:297-310, 1977).     

Purine metabolism in lymphocytes from patients with primary hypogammaglobulinaemia

The previous report of low levels of purine 5'-nucleotidase activity in peripheral blood mononuclear cells (lymphocytes and monocytes) from patients with non-familial adult onset `variable' primary hypogammaglobulinaemia has been confirmed and the observation extended to include patients with other types of primary immunodeficiency. Patients with sex-linked congenital hypogammaglobulinaemia have values for mononuclear cell 5'-nucleotidase activity which are in the normal range, whereas most cases of non-familial adult onset `variable' primary hypogammaglobulinaemia have clearly subnormal values. The three patients with isolated IgA deficiency who were tested also had subnormal values. Evidence that the measured enzyme activity is in fact 5'-nucleotidase and independent of interfering phosphatase activities is presented. No significant or consistent alterations in the activities of the following enzymes were detected in mononuclear cells or erythrocytes: adenosine deaminase, purine nucleoside (inosine) phosphorylase, hypoxanthine phosphoribosyltransferase, adenine phosphoribosyltransferase, phosphoribosylpyrophosphate (PRPP) synthetase. The erythrocyte PRPP content and the mononuclear cell PRPP amidotransferase activity were normal in the small number of patients in which they were measured. These findings are discussed in the light of the current interest in the inter-relationship between some disorders of purine metabolism and the immunological deficiency syndromes.     

Tissue adenosine content in active soleus and gracilis muscles of cats

Vascularly isolated cat soleus and gracilis muscles were stimulated to contract isometrically and were then frozen in situ. Adenosine, inosine, and hypoxanthine (nucleosides), and lactate were measured in neutralized, perchloric acid extracts of muscle. During contraction, nucleoside content increased in soleus muscle but changed little in gracilis muscle. However, adenosine content did not correlate with vascular conductance or oxygen consumption in either soleus or gracilis muscle. Adenosine content did correlate with lactate content in soleus but not gracilis muscle. The activity of AMP deaminase was highest in cat gracilis muscle and lowest in dog cardiac muscle. The activity of 5'-nucleotidase was lowest in cat gracilis muscle and highest in dog cardiac muscle. Cat soleus and dog gracilis muscles had intermediate activities of both enzymes. The findings of the present study do not support a role for adenosine in mediating prolonged active hyperemia in fast-twitch gracilis muscle of cats and cast doubt on such a role in slow-twitch soleus muscle of cats. Differences in the activities of AMP deaminase and 5'-nucleotidase provide a qualitative, biochemical explanation for apparent differences in net adenosine production among muscles composed of different fiber types and between skeletal and cardiac muscle.     

Adenosine A1 receptors in the human brain: a quantitative autoradiographic study

The distribution of adenosine A1 receptors in the human brain was studied by autoradiography in post mortem brain tissues from 26 subjects without reported neurological disease. N6-[3H]Cyclohexyl-adenosine was used as the ligand. For comparison, adjacent sections of some regions were examined histochemically for 5'-nucleotidase activity. The receptor sites were heterogeneously distributed throughout the CNS. The highest receptor densities were found in the stratum oriens, pyramidale and radiatum of the hippocampus. High densities were also found in the cerebral cortex and the striatum. In the thalamus there was a heterogeneous distribution of binding sites with a high density in structures such as the medial and anterior nucleus. Intermediate receptor densities were found in the accumbens, the olfactory tubercle and most parts of the amygdala among others. The hypothalamus had low receptor densities. In the brainstem and the spinal cord very low receptor concentrations were found. However, in some structures such as the substantia nigra, the colliculus superior and the substantia gelatinosa of the spinal cord a low level of binding could be measured. The cerebellar cortex showed low densities of receptors. Structures showing high levels of 5'-nucleotidase activity were the hippocampus, the striatum and parts of the cerebral cortex among other regions. In general there was a poor correlation between the localization of A1 receptors and the 5'-nucleotidase activity. Some regions, however, showed a similar distribution of these two markers. In general, the distribution of adenosine A1 receptors found in the human brain is comparable to that found in previous autoradiographic studies in the rat brain. However, some regional differences were observed in, for example, the cerebral cortex, the striatum and the cerebellar cortex. These differences may prove to be functionally relevant.     

5'-nucleotidase of B and T lymphocytes isolated from human peripheral blood.

The ecto-5'-nucleotidase activities of highly purified T and B lymphocytes from human peripheral blood have been investigated using biochemical and histochemical techniques. The enzyme activity of the purified B cells was about 3.5 times that of the T cells. Using a histochemical assay, 21--55% of the B cells stained positively for 5'-nucleotidase, but only 2--22% of the T cells were positive. These results are discussed in relation to the low 5'-nucleotidase activities found on peripheral blood lymphocytes from patients with chronic lymphatic leukaemia and some patients with primary hypogammaglobulinaemia.     

Semiautomatic method of measurement of serum 5'nucleotidase activity]

A semi-automated method for determining the 5'-nucleotidase activity in human serum for use with the Technicon Autoanalyzer I is described. Based upon the manual method of Persijn and Van Der Slik, adenosine formed by hydrolysis of 5'-AMP is deamined enzymatically and ammonia determined with the Berthelot reaction. The non specific alkaline phosphatases are inhibited by phenylphosphate. Concentrations of 5'-AMP and phenylphosphate are discussed. Nucleotidase activity is determined without dilution up to a value of 210 UI/liter. The precision is better than other manual or semi-automated methods in current use.     

Enzyme histochemistry and thyroid neoplasia

The authors have examined the enzyme histochemical staining of surgically removed human thyroid tissue in an attempt to identify markers that might be useful in the histopathologic diagnosis of thyroid neoplasms. Fresh thyroid glands and other tissues were fixed in cold (4 degrees C) 4% paraformaldehyde and embedded in glycol methacrylate. Forty-two specimens were studied in thin sections, which gave excellent histologic detail and enzyme preservation. Cytologic detail was similar to that in Papanicolaou-stained smears, with good definition of nuclear inclusions and grooves, particularly in cases of papillary carcinoma. The enzyme histochemical reactions studied were as follows: adenosine triphosphatase, alkaline and acid phosphatases, alpha-naphthyl acetate esterase, and 5'-nucleotidase. Thyroid epithelial cells and the benign neoplasms derived from them were typically positive for 5'-nucleotidase, alpha-naphthyl acetate esterase, and acid phosphatase, and negative for adenosine triphosphatase and alkaline phosphatase. Staining for adenosine triphosphatase was present in papillary and follicular carcinomas and was seen in benign glands only under certain circumstances such as Graves' disease. The adenosine triphosphatase reaction therefore appears to be helpful in distinguishing between benign and malignant neoplasms derived from thyroid epithelium in humans and may be a useful adjunct to routine morphology.     

The importance of B-cells and ecto-5'nucleotidase in Mycoplasma fermentans infection and the relevance to rheumatoid arthritis

The aim of this work was to discover if Mycoplasma fermentans, which is known to infect B cells, could be the cause of the raised ecto-5'-nucleotidase observed in the synovial fluid of rheumatoid arthritis patients. The ecto-5'-nucleotidase activity in the patients' serum has been shown to correlate with the erythrocyte sedimentation rate and DNA from the mycoplasma has been found in the synovial fluid. B lymphoblastoid cell lines were exposed to 16 strains of Mycoplasma fermentans and their ecto-5'-nucleotidase, CD73, was measured both biochemically and by mouse antibodies to human ecto 5'-nucleotidase using the fluorescence activated cell sorter. The type strain, PG 18, did not grow with the B cells. Some of the mycoplasma strains (9/15) increased the cellular ecto-5'-nucleotidase activity from twice to 17 fold, and usually showed 5'-nucleotidase activity themselves. At least one strain, M106, induced human 5'-nucleotidase on the normally 5'-nucleotidase negative Daudi and Raji Burkitt's lymphoma cell lines, and increased sevenfold the 5'-nucleotidase on the monocyte/macrophage cell line THP-1. Growing the cells in aged medium increased the level of mycoplasma infection. This mycoplasma-induced enzyme showed a conformational change and an increase in activity with a glycosylation change involving mannose groups. The other group of strains, mostly of respiratory or cell culture origin, usually did not have any 5'-nucleotidase of their own and decreased the B-cell enzyme activity by about half. Electron microscopy and flow cytometry showed that the strain M106 was filamentous and could be found inside the B-cells. The 5'-nucleotidase-inducing strains of M. fermentans may be important in the aetiology of rheumatoid arthritis.