Doris Kloor

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A2A adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (Treg) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4+ and CD8+ T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated Treg. Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4+ T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of Treg but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Role of extracellular nucleotide phosphohydrolysis in intestinal ischemia-reperfusion injury

Extracellular adenosine has been implicated as an innate antiinflammatory metabolite, particularly during conditions of limited oxygen availability such as ischemia. Because extracellular adenosine generation is primarily produced via phosphohydrolysis from its precursor molecule adenosine‐monophosphate (AMP) through the enzyme ecto‐5′‐nucleotidase (CD73), we examined the contribution of CD73‐dependent adenosine production in modulation of intestinal ischemia‐reperfusion (IR) injury. Following transcriptional and translational profiling of intestinal tissue that revealed a prominent induction of murine CD73, we next determined the role of CD73 in protection against intestinal IR injury. Interestingly, pharmacological inhibition or targeted gene deletion of CD73 significantly enhanced not only local intestinal injury, but also secondary organ injury, following IR as measured by intestinal and lung myeloperoxidase, aspartate and alanine aminotransferase, interleukin (IL) ‐1, IL‐6, and histological injury. To confirm the role of CD73 in intestinal adenosine production, we measured adenosine tissue levels and found that they were increased with IR injury. In contrast, CD73‐deficient (cd73−/−) mice had lower adenosine levels at baseline and no increase with IR injury. Finally, reconstitution of cd73−/− mice or treatment of wild‐type mice with soluble 5′‐nucleotidase was associated with significantly lower levels of injury. These data reveal a previously unrecognized role of CD73 in attenuating intestinal IR‐mediated injury.—Hart, M. L., Henn, M., Köhler, D., Kloor, D., Mittelbronn, M., Gorzolla, I. C., Stahl, G. L., Eltzschig, H. K. Role of extracellular nucleotide phosphohydrolysis in intestinal ischemia‐reperfusion injury. FASEB J. 22, 2784–2797 (2008)

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography.

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N6-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25-1.0 nmol/g wet weight for renal tissue and 0.25-0.5 microM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1 +/- 2.6% and 102.5 +/- 4.0% for renal tissue and urine, respectively (mean +/- S.D., n = 3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64 +/- 2.2, 0.67 +/- 0.18 and 46.2 +/- 1.9 nmol/g wet weight, respectively (mean +/- S.D., n = 6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low microM range and are negatively correlated with urine flow-rate.

Direct Treatment of Mouse or Human Blood With Soluble 5′-Nucleotidase Inhibits Platelet Aggregation

Objective—Adenosine signaling is known to inhibit platelet aggregation. Extracellular adenosine mainly stems from enzymatic phosphohydrolysis of precursor nucleotides via ecto-5′-nucleotidase. Previous studies suggest that soluble 5′-nucleotidase (5′-NT) derived from Crotalus atrox venom may be clinically beneficial in vascular leakage, myocardial, renal, and intestinal ischemia, or acute lung injury. However, the effects of 5′-NT treatment on platelet aggregation remain unknown. We examined the direct effects of 5′-NT treatment on platelet aggregation in vivo and ex vivo using a whole blood aggregation method. Methods and Results—Platelet aggregation in whole human blood was completely inhibited by 5′-NT. When 5′-[αβ-methylene] diphosphate (APCP), a specific 5′-ecto-nucleotidase inhibitor, was added together with 5′-NT, APCP fully restored collagen- or ADP-induced aggregation. Adenosine levels in whole blood were significantly increased after 5′-NT treatment compared to controls and inhibition of platelet aggregation by 5′-NT was completely reversed by pretreatment with the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline hydrate (8-SPT), suggesting that 5′-NT inhibits aggregation via increased adenosine signaling. Administration of 5′-NT to mice in vivo abolished ADP- and collagen-induced platelet aggregation and increased adenosine concentrations and tail bleeding time. Conclusions—5′-NT treatment inhibits platelet aggregation via generation of increased levels of extracellular adenosine and subsequent adenosine receptor signaling.

Involvement of Adenosine A2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble “find me” signals, 2) recognition and phagocytosis via cell surface-presenting “eat me” signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.

S-Adenosylhomocysteine-Hydrolase from Bovine Kidney: Enzymatic and Binding Properties

In the present study S-adenosylhomocysteine (SAH) hydrolase from the bovine kidney has been purified to apparent homogeneity by standard chromatographic procedures. The purified enzyme was free from adenosine deaminase activity and showed a one-banded pattern in SDS-PAGE with a monomer molecular mass of 47,500. The molecular mass of the native enzyme estimated by gel filtration was about 190,000. The pI was 5.5. For hydrolysis of SAH we found a Km of 5.0 +/- 1.2 microM and a V of 0.25 mumol/min/mg. In the direction of synthesis the Km for adenosine was 5.6 microM and V 0.53 mumol/min/mg. The enzyme activity was inhibited in the presence of adenosine with a Ki = 3 microM. In a second set of experiments we determined the binding characteristics of [3H]-adenosine to purified enzyme. The enzyme bound [3H]-adenosine with three apparent affinities: Kd1 = 6.8 +/- 0.7 nM and Bmax1 = 0.24 +/- 0.04 nmol/mg protein; Kd2 = 387 +/- 41 nM and Bmax2 = 1.4 nmol/mg protein, and Kd3 = 7.05 +/- 0.9 microM and Bmax3 = 9 nmol/mg protein. Binding of 25 nM [3H]-adenosine obeyed a monophasic reaction with a k+1 value of 0.025 min/nM. Dissociation of [3H]-adenosine-SAH hydrolase complex was markedly temperature dependent. After a 240-min incubation at 0 degrees C only 5-10% and at 20 degrees C 75% were displaceable. A fraction of 25% bound [3H]-adenosine was not displaceable by unlabeled adenosine. Our data show that the renal SAH hydrolase exhibits similar enzyme kinetics as the well-characterized SAH hydrolase from liver. The amount of SAH hydrolase present in renal tissues (1.4 nmol/g wet weight) could account almost entirely for the binding of renal tissue adenosine. Finally, we report for the first time a high affinity binding site of SAH hydrolase for adenosine, which remains unexplained at present.

Effects of ions on adenosine binding and enzyme activity of purified S-adenosylhomocysteine hydrolase from bovine kidney.

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.

Localization of S-adenosylhomocysteine Hydrolase in the Rat Kidney:

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 ± 0.02 mU/mg in the kidney, 0.32 ± 0.03 mU/mg in the glomeruli, and 0.50 ± 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation.

Adenosine binding sites at S-adenosylhomocysteine hydrolase are controlled by the NAD+/NADH ratio of the enzyme

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine. On the basis of the kinetics of Ado binding to AdoHcy hydrolase we have shown that AdoHcy hydrolase binds Ado with different affinities [Kidney Blood Press. Res. 19 (1996) 100]. Since AdoHcy hydrolase in its totally reduced form binds Ado with high affinity we determined in the present study the Ado binding characteristics of purified AdoHcy hydrolase from bovine kidney (native form) and of reconstituted forms with defined NAD(+)/NADH ratios. AdoHcy hydrolase in its native form and at a ratio of 50% NAD(+) and 50% NADH exhibits two binding sites for Ado with a K(D1) of 9.2+/-0.6 nmol/L and a K(D2) of 1.4+/-0.1 micromol/L, respectively. Binding of Ado to AdoHcy hydrolase in its NADH form and in its NAD(+) form exhibits only one binding site with high affinity 48.3+/-2.7 nmol/L for the NADH form and with a low affinity of 4.9+/-0.3 micromol/L for the NAD(+) form. To identify these two Ado binding sites, AdoHcy hydrolase was covalently modified with [2-3H]-8-azido-Ado. After irradiation of the native AdoHcy hydrolase two different photolabeled peptides were isolated and identified as Asp(307)-Val(325) and Tyr(379)-Thr(410). When the reconstituted AdoHcy hydrolase in its NADH and in its NAD(+) form was irradiated with [2-3H]-8-azido-Ado only one peptide was identified as Asn(312)-Lys(318) from the NADH form and as Asp(391)-Ala(396) from the NAD(+) form. Based on the crystallographic data, the labeled peptide Asp(391)-Ala(396) (low affinity binding site), appears to belong to the catalytic domain of AdoHcy hydrolase, whereas the labeled peptide, identified as Asn(312)-Lys(318) (high affinity binding site), is located in the NAD domain. In conclusion, our data show that AdoHcy hydrolase has two different Ado binding sites which are dependent upon the enzyme-bound NAD(+)/NADH ratios.

S-Adenosylhomocysteine Metabolism in Different Cell Lines: Effect of Hypoxia and Cell Density

Background/Aims: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. Methods: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. Results: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 ± 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 ± 1.1 and 21.1 ± 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 ± 0.7 and 7.1 ± 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 ± 0.2 nmol min-1 mg-1 protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. Conclusions: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.