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The role of plasma adenosine deaminase in chemoattractant-stimulated oxygen radical production in neutrophils
ARTICLE IN PRESS European Journal of Cell Biology 89 (2010) 462–467
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The role of plasma adenosine deaminase in chemoattractant-stimulated oxygen radical production in neutrophils a,c,n ¨ ¨ Hanna Kalvegren , Jonna Fridfeldt b, Torbjorn Bengtsson b,c a
Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Faculty of Health Sciences, Link¨ oping University, SE-581 85 Link¨ oping, Sweden Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Link¨ oping University, SE-581 85 Link¨ oping, Sweden c ¨ rebro University, SE-701 82 O ¨ rebro, Sweden Division of Clinical Medicine, Department of Biomedicine, O b
a r t i c l e in f o
a b s t r a c t
Article history: Received 11 June 2009 Received in revised form 18 November 2009 Accepted 18 December 2009
Objectives: Adenosine deaminase (ADA) has a role in many immunity mediated disorders, such as asthma, tuberculosis and coronary artery disease. This study aims to investigate the ability of plasma ADA to modulate reactive oxygen species (ROS) production in neutrophils, and examine the involvement of adenosine and the cyclic AMP signaling pathway in this process. Methods: Neutrophils were stimulated, in the absence or presence of plasma, with the chemotactic peptide fMLP (formyl-methionyl-leucyl-phenylalanine), and the ROS production was determined with luminol-enhanced chemiluminescence. Activity of ADA was measured spectrophotometrically. Results: Plasma dose-dependently amplified the ROS generation in fMLP-stimulated neutrophils. In parallel, incubation of neutrophils in plasma elevated the total ADA-activity approximately 10 times from 1.3 U/ml to 12 U/ml. Inhibition of ADA, or type IV phosphodiesterases, significantly lowered the plasma-mediated ROS production. Furthermore, the high-affinity adenosine A1 receptor antagonists DPCPX and 8-phenyltheophylline markedly inhibited the plasma-induced respiratory burst in neutrophils, suggesting an A1 receptor–mediated mechanism. Conclusions: This study suggests that plasma ADA amplifies the release of toxic oxygen radicals from neutrophils through a downregulation of the inhibitory adenosine/cAMP-system and an enhanced activation of the stimulatory adenosine A1-receptor. This mechanism has probably a crucial role in regulating neutrophil function and in the defence against microbial infections. However, a sustained neutrophil activation could also contribute to inflammatory disorders such as atherosclerosis. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Leukocyte Reactive oxygen species Adenosine deaminase Plasma Phosphodiesterase Inflammation Adenosine A1 receptor antagonist
Introduction Neutrophil granulocytes form the first line of defence against invading microorganisms and play a crucial role in the inflammatory reaction of various diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), asthma and atherosclerosis (Nathan, 2006). Chemotactic agents rapidly recruit circulating neutrophils to inflammatory sites where their activation leads to the release of bactericidal lysosomal enzymes and reactive oxygen species (ROS) (Nathan, 2006). Imbalance between activation and inhibition of these defence mechanisms may contribute to the development of inflammatory and vascular disorders (Valko et al., 2006). Accumulation of ROS at the vascular endothelium due to activated leukocytes might cause endothelial
n Corresponding author at: Division of Clinical Medicine, Department of ¨ rebro University, SE-701 82 O ¨ rebro, Sweden. Biomedicine, O Tel.: + 46 19 6026607; fax: + 46 19 6026650. ¨ E-mail address: [email protected] (H. Kalvegren).
0171-9335/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2009.12.004
cell dysfunction and initiate and propagate the atherosclerotic process (Lubos et al., 2008). Furthermore, leukocyte-derived superoxide anion interferes with nitric oxide, which is recognized as an anti-atherogenic molecule inhibiting leukocyte and platelet activation (Lubos et al., 2008). Adenosine is a signalling nucleoside that has potent antiinflammatory and tissue protective effects in acute and chronic injury processes (Eltzschig et al., 2008). The vascular endothelium constitutively liberates adenosine, which acts as a physiological defence mechanism against the harmful effects of uncontrolled activation of leukocytes and platelets. Adenosine inhibits neutrophil functions (e.g. adhesion to endothelial cells and ROS production) at micromolar concentrations by occupying the low affinity adenosine A2 receptor, whereas binding of adenosine at pico- to nanomolar concentrations to the high affinity A1 receptor stimulates neutrophil functions (Cronstein, 1994). The A2 receptor mediates adenylate cyclase activation and cAMP formation, whereas the A1 receptor inhibits adenylate cyclase activity (Hasko and Pacher, 2008). Cyclic AMP-mediated down regulation of the inflammatory response due to A2 receptor activation might
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be enhanced by blocking phosphodiesterase (PDE)-mediated hydrolysation of cAMP to 5AMP (Hasko and Pacher, 2008) with specific PDE-inhibitors. In order to adhere to the vessel wall and subsequently migrate into inflamed tissue and combat invading microbes, the neutrophil has to overcome the inhibitory effects of adenosine. Adenosine deaminase (ADA) is an intra- and extracellular enzyme involved in purine metabolism by catalyzing the hydrolytic deamination of adenosine to inosine (Yegutkin, 2008). Previous investigations indicate that an elevated activity and/or expression of ADA amplify the neutrophil responsiveness to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) (Cronstein et al., 1983, Bengtsson et al., 1996). Plasma ADA activity has been shown to increase significantly during different bacterial infections, and might thus be an important bystander of the inflammatory response of neutrophils (Tuon et al., 2006, Klockars et al., 1991). Several studies suggest that ADA has a crucial role in many inflammatory disorders, such as rheumatic diseases, COPD, intestinal inflammatory disorders and atherosclerosis by antagonizing the anti-inflammatory action of adenosine (Sari et al., 2003, Cavalcante et al., 2006, Sun et al., 2006, Antonioli et al., 2007, Chavan et al., 2007, Brown et al., 2008). In this study, we have examined the capacity of plasma ADA to modulate chemotactic peptide-induced ROS production in neutrophils, and evaluated the involvement of adenosine, adenosine receptors and the cAMP system. Materials and methods Chemicals and buffers Chemicals and their sources were as follows: 4-(3-butoxy-4metoxybenzyl)-2-imidazolidinone (RO 20-1724), 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), 3,7- dimethyl-1-propargylxanthine (DMPX), erythro-9-(-2-hydroxy-3-nonyl)-adenine (EHNA) (Research Biochemicals Int., Natick, MA, USA); adenosine deaminase (ADA), adenosine, 5-amino-2,3-dihydro-1,4-phtalazinedione (luminol), formyl-methionyl-leucyl-phenylalanine (fMLP), forskolin, horseradish peroxidase (HRP), superoxide dismutase (SOD), 8-phenyltheophylline, N6-Cyclopentyladenosine (CPA), 2-p-(2Carboxyethyl)phenethylamino-50 -N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS 21680), phenol, hypochlorite (Sigma Chemical Co., St Louis, MO, USA); catalase (Boehringer Mannheim, Mannheim, Germany); N-(2-((p-bromocinnamyl)amino)ethyl)-5isoqquinolinesulfonamide (H89) (Calbiochem, San Diego, CA, USA); phosphate-buffered saline (PBS) pH 7,3 [NaCl (137 mmol/L), KCL (2,7 mmol/L), NA2HPO4 2 H2O (6,7 mmol/L), KH2PO4 (1,5 mmol/L)]; Krebs-Ringer phosphate buffer (KRG) pH 7,3 [NaCl (120 mmol/L), KCl (4,9 mmol/L), MgSO4 7H2O (1,2 mmol/L), KH2PO4 (1,7 mmol/L), NA2HPO4 2H2O (8,3 mmol/L), Glukos (10 mmol/L), CaCl2 2H2O (1 mmol/L)]. Isolation of human granulocytes and preparation of platelet-rich plasma Neutrophil granulocytes (neutrophils) were isolated from heparinized human peripheral blood, donated by apparently healthy non-medicated adult volunteers at the blood bank at ¨ Linkoping University Hospital. Blood and neutrophils were handled using plastic utensils exclusively, and calcium was excluded in buffers during isolation. The neutrophils were prepared essentially as pioneered by ¨ Boyum (Boyum, 1968). A density separation medium was prepared by careful layering of one part of LymphoprepTM over four parts of PolymorphprepTM. Whole blood was layered on an
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equal volume of separation liquid and the tubes centrifuged in a swing-out rotor for 40 min (480 x g, room temperature), yielding an upper band containing mononuclear cells, a middle band containing neutrophils and a pellet of red blood cells. The neutrophils were harvested and the separation liquid removed by dilution in PBS and another centrifugation for 10 min at 480 x g. The remaining erythrocyte contamination was lysed by 35 s of exposure to ice-cold distilled water, followed by washing of the cells twice in KRG (200 x g at 4 1C). The neutrophils were counted in a Coulter Counter ZM Channelyser 256 (Coulter-Electronics Ltd., Luton, U.K.) and stored on ice. In some experiments, neutrophils were isolated in the presence of ADA. ADA was added to whole blood (0,27 units/ ml) and during the different steps in the preparation of neutrophils. Plasma was prepared from the heparinized whole blood (see above) by centrifugation at 220 g for 20 min at 22 1C (model CPR, Beckman, U.K). The upper layer was carefully removed and stored in room temperature until use. This is a gentle separation procedure of the plasma to minimize the risk for cellular activation and release of intracellular components. However, this procedure does not exclude platelets from the plasma. Chemiluminescence Neutrophils (5 105 cells/ml) and plasma were separately preincubated, with or without different chemicals, in 24-well plates (NUNC Brand products, Denmark) for 5 min at 37 1C under stirring conditions in a rotary shaker, before being mixed together in different ratios and incubated for another 2 min. The cell suspensions were then rapidly transferred to cuvettes (Sarstedt, Nurnbrecht, Germany) containing luminol (50 mmol/L) and horse radish peroxidase (HRP, 4 U/ml) stimulated with fMLP (10-7 mol/ L) and monitored for chemiluminescence (CL) at 37 1C in a sixchannel Biolumat (LB 9505C, Berthold Co.,Wildbaden, Germany). With these reagents, both intra- and extracellulary produced ROS are detected since luminol is membrane permeable and peroxidase is sufficiently available in both intra- and extracellular compartments. The intracellular ROS generation was registered after exchange of extra peroxidase for 200 U/mL superoxide dismutase and 2,000 U/mL catalase that scavenges extracellular superoxide anion and hydrogen peroxide, respectively. Adenosine deaminase activity Neutrophils (5x105 cell/ml) and plasma were separately in 24-well plates (Sarstedt, Newton, NC, USA) for 5 min in a preheated shaker (Wesbart IS89, Wesbart Ltd, England) before being mixed together and incubated for another 2 min. The samples were stimulated with fMLP (10-7 mol/L) for 1, 10 or 30 mins before the reactions were stopped by hypothermic conditions. Adenosine deaminase (ADA) activity was determined according to the method by Giusti and Galanti (1984) based on the Bertolet reaction. In brief, ammonia liberated from adenosine reacts with phenol and hypochlorite to form the coloured compound indophenol, which is measured spectrophotometrically at 620 nm. The absorbance of indophenol is proportional to the amount of ammonium. One unit of ADA is defined as the amount of enzyme required to liberate 1 micromole of ammonium per minute from adenosine. Statistics Data are expressed as the mean 7 SEM. Statistical differences between means were assessed by the paired, two tailed students
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t-test or one-way anova. Po0,05 was considered to be statistical significant.
Results Plasma prolongs the ROS production in fMLP-stimulated neutrophils We have used luminol-enhanced chemiluminescence (CL) to evaluate the effects of plasma on neutrophil oxidase activity. Exposure of human neutrophils to the chemotactic peptide fMLP (10-7 mol/L) caused a rapid CL response, peaking after 30 s and lasting for 10-15 min (Fig. 1). In a dose-dependent manner, plasma markedly inhibited the early release of oxygen metabolites, but caused an extensive secondary ROS production lasting for at least 45 min (Fig. 1). Incubation with the membrane impermeable O-2 and H2O2 scavangers, superoxide dismutase (200 U/mL) and catalase (2000 U/mL) respectively, markedly reduced the CL-response to fMLP in the neutrophil control (data not shown). SOD and catalase totally antagonized the effects of plasma on the fMLP-induced CL response, indicating that plasma primarily contributes to an extracellular release of ROS from neutrophils (Fig. 1). The ROS production is dependent on ADA activity We and others have previously demonstrated that adenosine is a potent modulator of neutrophil oxidase activity and might thus be involved in the plasma-induced ROS production. Consequently, we examined whether removal of extracellular adenosine by plasma ADA has a role in the stimulatory effects of plasma on neutrophil oxidase activity. We found that the ADA inhibitor EHNA (10 mmol/L) totally obstructed the plasma-mediated enhancement of the fMLP-induced ROS production (Fig. 2B), while EHNA had almost no effect on the CL response in the neutrophil control (Fig. 2A). Introduction of ADA (0.27 U/mL) in the neutrophil control increased the fMLP-induced ROS production
Fig. 2. Effects of adenosine/cAMP modulators on fMLP-stimulated neutrophil ROSproduction in the presence or absence of plasma. Experimental conditions were as in Fig. 1. The cell suspensions were pre-treated with ADA (0,27 units/ml), the ADA inhibitor EHNA (10 mmol/L), the phosphodiesterase inhibitor RO 20-1724 (10 mmol/L) or the adenylate cyclase stimulator forskolin (10 mmol/L), for 7 min, followed by stimulation with fMLP(10-7 mol/L) and chemiluminescence (CL) measurement in the absence or presence of plasma (10%). (A) The CL-response from fMLP-stimulated neutrophils incubated with ADA or the different inhibitors in percent of a fMLP-stimulated neutrophil control. (B) The CL-response from a fMLP-stimulated neutrophil/plasma mixture incubated with ADA or the different inhibitors in percent of a fMLP-stimulated neutrophil/plasma control. The data represents the mean 7 SEM of 3-7 separate experiments.
Fig. 1. The effect of plasma on neutrophil oxygen radical production. Neutrophils (5x105 /ml) and plasma were separately incubated for 5 min at 37 1C under stirring conditions in 6-well plates and then mixed (v/v) and incubated for another 2 min. The neutrophil control was incubated for 7 min under the same conditions. The samples were thereafter monitored for luminol-amplified chemiluminescence (CL) during stimulation with fMLP (10-7 mol/L). The figure shows the CL-curve of neutrophil incubated in the absence (control) or presence of different amounts of plasma (the amount of plasma is shown as percent of the total volume after added to the cell suspension). The CL-response of unstimulated plasma 10%, and a fMLP (10-7 M)-stimulated neutrophil/plasma (10%) mixture incubated with SOD and catalase is also shown. The curves show representative recordings of 5-10 separate experiments.
by approximately 20 % (not statistically significant, Fig. 2A). In contrast, exogenous ADA had no effects on the fMLP-triggered ROS production in neutrophils incubated with plasma (Fig. 2B).
Activity of ADA The ADA activity in neutrophil and plasma suspensions was analyzed spectrophotometrically (Giusti and Galanti, 1984).
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We found that a washed neutrophil suspension expressed an ADA activity of approximately 1.3 U/mL (Fig. 3). Plasma contributed with a marked elevation of the ADA activity when added to the neutrophil suspension. The ADA activity was approximately 11 U/mL in plasma and 12 U/mL in a neutrophil-plasma mixture (Fig. 3). The ADA activity in the neutrophil-plasma mixture slightly increased 10 and 30 min after incubation (Fig. 3).
Inhibition of the adenosine A1 receptors reduces the ROS production To further clarify the role of adenosine in plasma, different adenosine receptor antagonists were tested. The A2 receptor antagonist DMPX (0.001-10 mmol/L) had no effects on the fMLP-
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stimulated neutrophils incubated with plasma (not shown). On the other hand, we found that the A1 receptor antagonists DPCPX and 8-phenyltheophylline, respectively, significantly inhibited the plasma-mediated enhancement of neutrophil ROS production (Fig. 4A). Extraordinary low doses of DPCPX and 8-phenyltheophylline were required to observe an inhibitory effect, and maximal inhibition (40 %) was obtained at 10-11 mol/L with both 8-phenyltheophylline and DPCPX (Fig. 4A). Adenosine A1 and A2 receptor agonists To further study the importance of the A1 receptor in neutrophil ROS production, we incubated the neutrophils with the A1 receptor agonist CPA prior to fMLP stimulation. Interestingly, CPA potentiated the fMLP-induced neutrophil ROS-production (Fig. 4B). CPA increased the ROS-response in neutrophils from all blood donors, but the extent of potentiation varied and thereby there were no statistical differences compared to neutrophil control. The A1 receptor agonist had no potentiating effect on fMLP-stimulated neutrophils incubated with plasma (Fig. 4B). In opposite, the A2 receptor agonist CGS 21680 significantly lowered the fMLP-induced ROS- response in neutrophils incubated with plasma (Fig. 4B). Inhibition of phosphodiesterase abolishes the ROS production
Fig. 3. Neutrophils (5x105 cell/ml) and plasma were separately preincubated in 24-well plates for 5 min in 37 1C before being mixed together and incubated for another 2 min. The samples were stimulated with fMLP (10-7 mol/L) for 1, 10 or 30 min before the reactions were stopped by hypothermic conditions.Adenosine deaminase (ADA) activity was determined according to the method by Giusti and Galanti (1984) (see Methods).
The role of the cAMP/protein kinase A pathway in the plasmamediated enhancement of neutrophil oxidase activity was evaluated by treating the neutrophils with the protein kinase A inhibitor H89, the adenylate cyclase activator forskolin, and the phosphodiesterase inhibitor RO 20-1724. Treatment with H89
Fig. 4. (A) Effects of two adenosine A1 receptor antagonists on ROS production in fMLP-stimulated neutrophils in the presence of plasma. Experimental conditions were as in Fig. 1. Neutrophils were preincubated with the A1 receptor antagonists (DPCPX) or 8-phenyltheophylline for 7 min, stimulated with fMLP (10-7 mol/L), and monitored for chemiluminescence (CL) in presence of plasma (10%). The data are based on the mean 7 SEM of 3-6 separate experiments. (B) Effect of A1 and A2 receptor agonists. Experimental conditions were as in Fig. 1. The A1 receptor agonist CPA or the A2 receptor agonist CGS 21680 were added to neutrophils or neutrophils incubated with plasma followed by fMLP stimulation (10-7 mol/L). The ROS production was registered by measuring chemiluminescence. The data are shown as percent of fMLPstimulated neutrophil control or fMLP- stimulated neutrophil/plasma control. The data are based on the mean 7 SEM of 3-6 separate experiments.
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(10 mmol/L), which previously has been shown to reverse inhibitory effects of adenosine, did not affect the plasmamediated ROS production in neutrophils (not shown). Stimulation of adenylate cyclase with forskolin (10 mmol/L) reduced the ROS production in the neutrophil control (Fig. 2A), whereas no effects were obtained on the CL response in neutrophils incubated with plasma (Fig. 2B). However, RO 20-1724 (10 mmol/L) totally abolished the plasma-mediated elevation of the fMLP-triggered ROS production (Fig. 2B), whereas the PDE-inhibitor had no significant effects on the fMLP-induced CL response of the neutrophil control (Fig. 2A).
Discussion Extracellular adenosine has a central role in the modulation of acute and chronic inflammation, e.g. by regulating the activity of neutrophil granulocytes (Cronstein, 1994, Hasko and Pacher, 2008). The ability of adenosine to exert pro- or anti-inflammatory effects is highly dependent on the balance between production and breakdown of this nucleoside. Removal of adenosine through deamination by ADA is considered to support pro-inflammatory processes (Yegutkin, 2008, Eltzschig et al., 2006). In this study, we demonstrate that an increased expression of ADA through neutrophil incubation in plasma markedly elevate the chemotactic peptide-induced generation of oxygen radicals. Plasma contains both inhibitors and activators of inflammatory reactions e.g. fibrinogen, albumin, IgG, adenine nucleotides and components of the complement, coagulation, kallikrein-kinin and fribinolysis systems (Cronstein, 1994). We used a gentle separation procedure that does not exclude platelets but minimize the risk for cellular activation. We have unpublished results showing no differences regarding the effect of platelet free plasma and plasma with platelets on neutrophil ROS production, which indicates no involvement of platelets. In the present study, we found a high expression of ADA in plasma. ADA catalyzes the hydrolytic deamination of adenosine to inosine and thereby lowers the concentration of adenosine and counteracts the inhibitory effects of this nucleoside. Our results show that the prolonged ROS production from neutrophils incubated in plasma was completely abolished by an ADA-inhibitor. In addition, the amplified neutrophil ROS production in plasma was not further enhanced by adding ADA, while exogenous ADA stimulated the ROS production in the neutrophil control. This supports that ADA expressed in plasma stimulates neutrophil ROS production induced by fMLP. Several studies have suggested an important regulatory function of ADA in plasma and in blood cells during different infectious and cardiovascular diseases (Chavan et al., 2007, Klockars et al., 1991). At low picomolar concentrations, adenosine binds to the A1 receptor leading to potentiated neutrophil activation. Ciruela et al. demonstrated on smooth muscle cells a close interaction between ADA and the A1 receptor, which modulates binding of adenosine and the associated intracellular signalling (Ciruela et al., 1996). In the present study, we demonstrate that A1 receptor antagonists significantly inhibit the fMLP-induced CL response in neutrophils incubated with plasma. This is interesting because pharmacological manipulations of adenosine and its metabolites have predominantly affected actions mediated via A2 receptors. Very low concentration of the A1 receptor antagonists was required for an inhibitory effect. This can possibly be explained by some unspecific effects of the A1 receptor antagonists at higher concentrations. The potentiating effect of the A1 receptor agonist CPA on the fMLP-induced neutrophil ROS-response further supports a triggering effect of this receptor. On the contrary, when the neutrophils were incubated with plasma there was no
stimulatory effect of the A1 receptor agonist, which indicates that this receptor is already activated under these conditions. Taken together, these results suggest that plasma ADA lowers the concentration of adenosine which leads to adenosine A1 receptor activation and an elevated oxygen radical production in neutrophils. Furthermore, Saura et al. have shown that ADA plays a key role in the regulation of A1 receptors by accelerating ligand induced receptor desensitisation and internalisation, and that the two cell surface proteins are internalised via the same endocytic pathway (Saura et al., 1998). In support, we found that the plasma-mediated effects on neutrophil oxidase activity decreased when neutrophils were isolated from blood in the presence of ADA, which might be due to a prior down regulation of A1 receptors from the neutrophil surface. There are a number of A1 receptor agonist and antagonists that are developed for clinical application in a variety of cardiovascular disorders (Hayes, 2003, Slawsky and Givertz, 2009). Furthermore, studies have shown that adenosine A1 receptor antagonists reduce renal impairment in patients with acute heart failure (Cotter et al., 2008) and bronchoconstriction and airway inflammation in astma (Nadeem et al., 2006). It is well known that oxidative stress is an important risk factor in atherosclerosis. Consequently, the decreased neutrophil ROS production by the adenosine A1 receptor antagonists shown in this study, could explain some of the beneficial effects of the antagonists in cardiovascular disease. An elevation of cAMP, triggered by endothelial-derived adenosine or prostaglandin, is considered to turn off neutrophil activities, and thereby antagonize the harmful effects of hydrolytic enzymes and toxic oxygen radicals in the vascular space (Ottonello et al., 1995). However, during the initial phases of neutrophil activation, including transmigration through layers of platelets and endothelial cells, an elevated PDE-activity is probably needed to counteract the inhibitory effects of cAMP (Lorenowicz et al., 2007). In this study, inhibition of class IV PDEs totally abolished the plasma-mediated ROS production in neutrophils. The plasma-induced second phase of neutrophil oxidase activity was not reduced when stimulating adenylate cyclase with forskolin, which moreover confirms a high PDE activity. Furthermore, inhibition of the cAMP-dependent protein kinase A did not affect the plasma-mediated enhancement of the fMLPtriggered ROS production, indicating that the inhibitory cAMP/PKA pathway is inactive. There are by now a large amount of evidence showing that leukocytes are involved in the development of vascular diseases and that oxygen radicals have a vital role (Weber et al., 2008, Cave et al., 2006, Quinn et al., 2008). The pro-inflammatory properties of plasma, including an enhanced and prolonged ROS-release, are probably important in the defence against pathogens. However, an excessive release of oxygen radicals in the vascular space might cause persistent inflammatory processes leading to arteriosclerosis (Loscalzo, 2003). It is considered that cAMP-elevation, induced by adenosine and A2 receptor activation, is part of an endogenous mechanism which down-regulates inflammatory reactions and the progression to chronic inflammation and tissue destruction (Lorenowicz et al., 2007). This study suggests that plasma increase the activity of ADA in bacterial peptidestimulated neutrophils, and thereby counteract an adenosineinduced autoregulatory inhibitory pathway. This probably facilitates the subsequent steps in the neutrophil activation, including the emigration from the vascular compartment to the inflamed extravascular tissue. However, an overexpressed ADA-activity antagonizing the anti-inflammatory effects of adenosine may contribute to chronic inflammation. Consequently, ADA inhibition may serve as therapeutic strategy in the treatment of inflammatory disorders, such as atherosclerosis and rheumatic diseases.
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European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb
The role of plasma adenosine deaminase in chemoattractant-stimulated oxygen radical production in neutrophils a,c,n ¨ ¨ Hanna Kalvegren , Jonna Fridfeldt b, Torbjorn Bengtsson b,c a
Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Faculty of Health Sciences, Link¨ oping University, SE-581 85 Link¨ oping, Sweden Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Link¨ oping University, SE-581 85 Link¨ oping, Sweden c ¨ rebro University, SE-701 82 O ¨ rebro, Sweden Division of Clinical Medicine, Department of Biomedicine, O b
a r t i c l e in f o
a b s t r a c t
Article history: Received 11 June 2009 Received in revised form 18 November 2009 Accepted 18 December 2009
Objectives: Adenosine deaminase (ADA) has a role in many immunity mediated disorders, such as asthma, tuberculosis and coronary artery disease. This study aims to investigate the ability of plasma ADA to modulate reactive oxygen species (ROS) production in neutrophils, and examine the involvement of adenosine and the cyclic AMP signaling pathway in this process. Methods: Neutrophils were stimulated, in the absence or presence of plasma, with the chemotactic peptide fMLP (formyl-methionyl-leucyl-phenylalanine), and the ROS production was determined with luminol-enhanced chemiluminescence. Activity of ADA was measured spectrophotometrically. Results: Plasma dose-dependently amplified the ROS generation in fMLP-stimulated neutrophils. In parallel, incubation of neutrophils in plasma elevated the total ADA-activity approximately 10 times from 1.3 U/ml to 12 U/ml. Inhibition of ADA, or type IV phosphodiesterases, significantly lowered the plasma-mediated ROS production. Furthermore, the high-affinity adenosine A1 receptor antagonists DPCPX and 8-phenyltheophylline markedly inhibited the plasma-induced respiratory burst in neutrophils, suggesting an A1 receptor–mediated mechanism. Conclusions: This study suggests that plasma ADA amplifies the release of toxic oxygen radicals from neutrophils through a downregulation of the inhibitory adenosine/cAMP-system and an enhanced activation of the stimulatory adenosine A1-receptor. This mechanism has probably a crucial role in regulating neutrophil function and in the defence against microbial infections. However, a sustained neutrophil activation could also contribute to inflammatory disorders such as atherosclerosis. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Leukocyte Reactive oxygen species Adenosine deaminase Plasma Phosphodiesterase Inflammation Adenosine A1 receptor antagonist
Introduction Neutrophil granulocytes form the first line of defence against invading microorganisms and play a crucial role in the inflammatory reaction of various diseases such as rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), asthma and atherosclerosis (Nathan, 2006). Chemotactic agents rapidly recruit circulating neutrophils to inflammatory sites where their activation leads to the release of bactericidal lysosomal enzymes and reactive oxygen species (ROS) (Nathan, 2006). Imbalance between activation and inhibition of these defence mechanisms may contribute to the development of inflammatory and vascular disorders (Valko et al., 2006). Accumulation of ROS at the vascular endothelium due to activated leukocytes might cause endothelial
n Corresponding author at: Division of Clinical Medicine, Department of ¨ rebro University, SE-701 82 O ¨ rebro, Sweden. Biomedicine, O Tel.: + 46 19 6026607; fax: + 46 19 6026650. ¨ E-mail address: [email protected] (H. Kalvegren).
0171-9335/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2009.12.004
cell dysfunction and initiate and propagate the atherosclerotic process (Lubos et al., 2008). Furthermore, leukocyte-derived superoxide anion interferes with nitric oxide, which is recognized as an anti-atherogenic molecule inhibiting leukocyte and platelet activation (Lubos et al., 2008). Adenosine is a signalling nucleoside that has potent antiinflammatory and tissue protective effects in acute and chronic injury processes (Eltzschig et al., 2008). The vascular endothelium constitutively liberates adenosine, which acts as a physiological defence mechanism against the harmful effects of uncontrolled activation of leukocytes and platelets. Adenosine inhibits neutrophil functions (e.g. adhesion to endothelial cells and ROS production) at micromolar concentrations by occupying the low affinity adenosine A2 receptor, whereas binding of adenosine at pico- to nanomolar concentrations to the high affinity A1 receptor stimulates neutrophil functions (Cronstein, 1994). The A2 receptor mediates adenylate cyclase activation and cAMP formation, whereas the A1 receptor inhibits adenylate cyclase activity (Hasko and Pacher, 2008). Cyclic AMP-mediated down regulation of the inflammatory response due to A2 receptor activation might
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be enhanced by blocking phosphodiesterase (PDE)-mediated hydrolysation of cAMP to 5AMP (Hasko and Pacher, 2008) with specific PDE-inhibitors. In order to adhere to the vessel wall and subsequently migrate into inflamed tissue and combat invading microbes, the neutrophil has to overcome the inhibitory effects of adenosine. Adenosine deaminase (ADA) is an intra- and extracellular enzyme involved in purine metabolism by catalyzing the hydrolytic deamination of adenosine to inosine (Yegutkin, 2008). Previous investigations indicate that an elevated activity and/or expression of ADA amplify the neutrophil responsiveness to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) (Cronstein et al., 1983, Bengtsson et al., 1996). Plasma ADA activity has been shown to increase significantly during different bacterial infections, and might thus be an important bystander of the inflammatory response of neutrophils (Tuon et al., 2006, Klockars et al., 1991). Several studies suggest that ADA has a crucial role in many inflammatory disorders, such as rheumatic diseases, COPD, intestinal inflammatory disorders and atherosclerosis by antagonizing the anti-inflammatory action of adenosine (Sari et al., 2003, Cavalcante et al., 2006, Sun et al., 2006, Antonioli et al., 2007, Chavan et al., 2007, Brown et al., 2008). In this study, we have examined the capacity of plasma ADA to modulate chemotactic peptide-induced ROS production in neutrophils, and evaluated the involvement of adenosine, adenosine receptors and the cAMP system. Materials and methods Chemicals and buffers Chemicals and their sources were as follows: 4-(3-butoxy-4metoxybenzyl)-2-imidazolidinone (RO 20-1724), 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), 3,7- dimethyl-1-propargylxanthine (DMPX), erythro-9-(-2-hydroxy-3-nonyl)-adenine (EHNA) (Research Biochemicals Int., Natick, MA, USA); adenosine deaminase (ADA), adenosine, 5-amino-2,3-dihydro-1,4-phtalazinedione (luminol), formyl-methionyl-leucyl-phenylalanine (fMLP), forskolin, horseradish peroxidase (HRP), superoxide dismutase (SOD), 8-phenyltheophylline, N6-Cyclopentyladenosine (CPA), 2-p-(2Carboxyethyl)phenethylamino-50 -N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS 21680), phenol, hypochlorite (Sigma Chemical Co., St Louis, MO, USA); catalase (Boehringer Mannheim, Mannheim, Germany); N-(2-((p-bromocinnamyl)amino)ethyl)-5isoqquinolinesulfonamide (H89) (Calbiochem, San Diego, CA, USA); phosphate-buffered saline (PBS) pH 7,3 [NaCl (137 mmol/L), KCL (2,7 mmol/L), NA2HPO4 2 H2O (6,7 mmol/L), KH2PO4 (1,5 mmol/L)]; Krebs-Ringer phosphate buffer (KRG) pH 7,3 [NaCl (120 mmol/L), KCl (4,9 mmol/L), MgSO4 7H2O (1,2 mmol/L), KH2PO4 (1,7 mmol/L), NA2HPO4 2H2O (8,3 mmol/L), Glukos (10 mmol/L), CaCl2 2H2O (1 mmol/L)]. Isolation of human granulocytes and preparation of platelet-rich plasma Neutrophil granulocytes (neutrophils) were isolated from heparinized human peripheral blood, donated by apparently healthy non-medicated adult volunteers at the blood bank at ¨ Linkoping University Hospital. Blood and neutrophils were handled using plastic utensils exclusively, and calcium was excluded in buffers during isolation. The neutrophils were prepared essentially as pioneered by ¨ Boyum (Boyum, 1968). A density separation medium was prepared by careful layering of one part of LymphoprepTM over four parts of PolymorphprepTM. Whole blood was layered on an
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equal volume of separation liquid and the tubes centrifuged in a swing-out rotor for 40 min (480 x g, room temperature), yielding an upper band containing mononuclear cells, a middle band containing neutrophils and a pellet of red blood cells. The neutrophils were harvested and the separation liquid removed by dilution in PBS and another centrifugation for 10 min at 480 x g. The remaining erythrocyte contamination was lysed by 35 s of exposure to ice-cold distilled water, followed by washing of the cells twice in KRG (200 x g at 4 1C). The neutrophils were counted in a Coulter Counter ZM Channelyser 256 (Coulter-Electronics Ltd., Luton, U.K.) and stored on ice. In some experiments, neutrophils were isolated in the presence of ADA. ADA was added to whole blood (0,27 units/ ml) and during the different steps in the preparation of neutrophils. Plasma was prepared from the heparinized whole blood (see above) by centrifugation at 220 g for 20 min at 22 1C (model CPR, Beckman, U.K). The upper layer was carefully removed and stored in room temperature until use. This is a gentle separation procedure of the plasma to minimize the risk for cellular activation and release of intracellular components. However, this procedure does not exclude platelets from the plasma. Chemiluminescence Neutrophils (5 105 cells/ml) and plasma were separately preincubated, with or without different chemicals, in 24-well plates (NUNC Brand products, Denmark) for 5 min at 37 1C under stirring conditions in a rotary shaker, before being mixed together in different ratios and incubated for another 2 min. The cell suspensions were then rapidly transferred to cuvettes (Sarstedt, Nurnbrecht, Germany) containing luminol (50 mmol/L) and horse radish peroxidase (HRP, 4 U/ml) stimulated with fMLP (10-7 mol/ L) and monitored for chemiluminescence (CL) at 37 1C in a sixchannel Biolumat (LB 9505C, Berthold Co.,Wildbaden, Germany). With these reagents, both intra- and extracellulary produced ROS are detected since luminol is membrane permeable and peroxidase is sufficiently available in both intra- and extracellular compartments. The intracellular ROS generation was registered after exchange of extra peroxidase for 200 U/mL superoxide dismutase and 2,000 U/mL catalase that scavenges extracellular superoxide anion and hydrogen peroxide, respectively. Adenosine deaminase activity Neutrophils (5x105 cell/ml) and plasma were separately in 24-well plates (Sarstedt, Newton, NC, USA) for 5 min in a preheated shaker (Wesbart IS89, Wesbart Ltd, England) before being mixed together and incubated for another 2 min. The samples were stimulated with fMLP (10-7 mol/L) for 1, 10 or 30 mins before the reactions were stopped by hypothermic conditions. Adenosine deaminase (ADA) activity was determined according to the method by Giusti and Galanti (1984) based on the Bertolet reaction. In brief, ammonia liberated from adenosine reacts with phenol and hypochlorite to form the coloured compound indophenol, which is measured spectrophotometrically at 620 nm. The absorbance of indophenol is proportional to the amount of ammonium. One unit of ADA is defined as the amount of enzyme required to liberate 1 micromole of ammonium per minute from adenosine. Statistics Data are expressed as the mean 7 SEM. Statistical differences between means were assessed by the paired, two tailed students
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t-test or one-way anova. Po0,05 was considered to be statistical significant.
Results Plasma prolongs the ROS production in fMLP-stimulated neutrophils We have used luminol-enhanced chemiluminescence (CL) to evaluate the effects of plasma on neutrophil oxidase activity. Exposure of human neutrophils to the chemotactic peptide fMLP (10-7 mol/L) caused a rapid CL response, peaking after 30 s and lasting for 10-15 min (Fig. 1). In a dose-dependent manner, plasma markedly inhibited the early release of oxygen metabolites, but caused an extensive secondary ROS production lasting for at least 45 min (Fig. 1). Incubation with the membrane impermeable O-2 and H2O2 scavangers, superoxide dismutase (200 U/mL) and catalase (2000 U/mL) respectively, markedly reduced the CL-response to fMLP in the neutrophil control (data not shown). SOD and catalase totally antagonized the effects of plasma on the fMLP-induced CL response, indicating that plasma primarily contributes to an extracellular release of ROS from neutrophils (Fig. 1). The ROS production is dependent on ADA activity We and others have previously demonstrated that adenosine is a potent modulator of neutrophil oxidase activity and might thus be involved in the plasma-induced ROS production. Consequently, we examined whether removal of extracellular adenosine by plasma ADA has a role in the stimulatory effects of plasma on neutrophil oxidase activity. We found that the ADA inhibitor EHNA (10 mmol/L) totally obstructed the plasma-mediated enhancement of the fMLP-induced ROS production (Fig. 2B), while EHNA had almost no effect on the CL response in the neutrophil control (Fig. 2A). Introduction of ADA (0.27 U/mL) in the neutrophil control increased the fMLP-induced ROS production
Fig. 2. Effects of adenosine/cAMP modulators on fMLP-stimulated neutrophil ROSproduction in the presence or absence of plasma. Experimental conditions were as in Fig. 1. The cell suspensions were pre-treated with ADA (0,27 units/ml), the ADA inhibitor EHNA (10 mmol/L), the phosphodiesterase inhibitor RO 20-1724 (10 mmol/L) or the adenylate cyclase stimulator forskolin (10 mmol/L), for 7 min, followed by stimulation with fMLP(10-7 mol/L) and chemiluminescence (CL) measurement in the absence or presence of plasma (10%). (A) The CL-response from fMLP-stimulated neutrophils incubated with ADA or the different inhibitors in percent of a fMLP-stimulated neutrophil control. (B) The CL-response from a fMLP-stimulated neutrophil/plasma mixture incubated with ADA or the different inhibitors in percent of a fMLP-stimulated neutrophil/plasma control. The data represents the mean 7 SEM of 3-7 separate experiments.
Fig. 1. The effect of plasma on neutrophil oxygen radical production. Neutrophils (5x105 /ml) and plasma were separately incubated for 5 min at 37 1C under stirring conditions in 6-well plates and then mixed (v/v) and incubated for another 2 min. The neutrophil control was incubated for 7 min under the same conditions. The samples were thereafter monitored for luminol-amplified chemiluminescence (CL) during stimulation with fMLP (10-7 mol/L). The figure shows the CL-curve of neutrophil incubated in the absence (control) or presence of different amounts of plasma (the amount of plasma is shown as percent of the total volume after added to the cell suspension). The CL-response of unstimulated plasma 10%, and a fMLP (10-7 M)-stimulated neutrophil/plasma (10%) mixture incubated with SOD and catalase is also shown. The curves show representative recordings of 5-10 separate experiments.
by approximately 20 % (not statistically significant, Fig. 2A). In contrast, exogenous ADA had no effects on the fMLP-triggered ROS production in neutrophils incubated with plasma (Fig. 2B).
Activity of ADA The ADA activity in neutrophil and plasma suspensions was analyzed spectrophotometrically (Giusti and Galanti, 1984).
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We found that a washed neutrophil suspension expressed an ADA activity of approximately 1.3 U/mL (Fig. 3). Plasma contributed with a marked elevation of the ADA activity when added to the neutrophil suspension. The ADA activity was approximately 11 U/mL in plasma and 12 U/mL in a neutrophil-plasma mixture (Fig. 3). The ADA activity in the neutrophil-plasma mixture slightly increased 10 and 30 min after incubation (Fig. 3).
Inhibition of the adenosine A1 receptors reduces the ROS production To further clarify the role of adenosine in plasma, different adenosine receptor antagonists were tested. The A2 receptor antagonist DMPX (0.001-10 mmol/L) had no effects on the fMLP-
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stimulated neutrophils incubated with plasma (not shown). On the other hand, we found that the A1 receptor antagonists DPCPX and 8-phenyltheophylline, respectively, significantly inhibited the plasma-mediated enhancement of neutrophil ROS production (Fig. 4A). Extraordinary low doses of DPCPX and 8-phenyltheophylline were required to observe an inhibitory effect, and maximal inhibition (40 %) was obtained at 10-11 mol/L with both 8-phenyltheophylline and DPCPX (Fig. 4A). Adenosine A1 and A2 receptor agonists To further study the importance of the A1 receptor in neutrophil ROS production, we incubated the neutrophils with the A1 receptor agonist CPA prior to fMLP stimulation. Interestingly, CPA potentiated the fMLP-induced neutrophil ROS-production (Fig. 4B). CPA increased the ROS-response in neutrophils from all blood donors, but the extent of potentiation varied and thereby there were no statistical differences compared to neutrophil control. The A1 receptor agonist had no potentiating effect on fMLP-stimulated neutrophils incubated with plasma (Fig. 4B). In opposite, the A2 receptor agonist CGS 21680 significantly lowered the fMLP-induced ROS- response in neutrophils incubated with plasma (Fig. 4B). Inhibition of phosphodiesterase abolishes the ROS production
Fig. 3. Neutrophils (5x105 cell/ml) and plasma were separately preincubated in 24-well plates for 5 min in 37 1C before being mixed together and incubated for another 2 min. The samples were stimulated with fMLP (10-7 mol/L) for 1, 10 or 30 min before the reactions were stopped by hypothermic conditions.Adenosine deaminase (ADA) activity was determined according to the method by Giusti and Galanti (1984) (see Methods).
The role of the cAMP/protein kinase A pathway in the plasmamediated enhancement of neutrophil oxidase activity was evaluated by treating the neutrophils with the protein kinase A inhibitor H89, the adenylate cyclase activator forskolin, and the phosphodiesterase inhibitor RO 20-1724. Treatment with H89
Fig. 4. (A) Effects of two adenosine A1 receptor antagonists on ROS production in fMLP-stimulated neutrophils in the presence of plasma. Experimental conditions were as in Fig. 1. Neutrophils were preincubated with the A1 receptor antagonists (DPCPX) or 8-phenyltheophylline for 7 min, stimulated with fMLP (10-7 mol/L), and monitored for chemiluminescence (CL) in presence of plasma (10%). The data are based on the mean 7 SEM of 3-6 separate experiments. (B) Effect of A1 and A2 receptor agonists. Experimental conditions were as in Fig. 1. The A1 receptor agonist CPA or the A2 receptor agonist CGS 21680 were added to neutrophils or neutrophils incubated with plasma followed by fMLP stimulation (10-7 mol/L). The ROS production was registered by measuring chemiluminescence. The data are shown as percent of fMLPstimulated neutrophil control or fMLP- stimulated neutrophil/plasma control. The data are based on the mean 7 SEM of 3-6 separate experiments.
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(10 mmol/L), which previously has been shown to reverse inhibitory effects of adenosine, did not affect the plasmamediated ROS production in neutrophils (not shown). Stimulation of adenylate cyclase with forskolin (10 mmol/L) reduced the ROS production in the neutrophil control (Fig. 2A), whereas no effects were obtained on the CL response in neutrophils incubated with plasma (Fig. 2B). However, RO 20-1724 (10 mmol/L) totally abolished the plasma-mediated elevation of the fMLP-triggered ROS production (Fig. 2B), whereas the PDE-inhibitor had no significant effects on the fMLP-induced CL response of the neutrophil control (Fig. 2A).
Discussion Extracellular adenosine has a central role in the modulation of acute and chronic inflammation, e.g. by regulating the activity of neutrophil granulocytes (Cronstein, 1994, Hasko and Pacher, 2008). The ability of adenosine to exert pro- or anti-inflammatory effects is highly dependent on the balance between production and breakdown of this nucleoside. Removal of adenosine through deamination by ADA is considered to support pro-inflammatory processes (Yegutkin, 2008, Eltzschig et al., 2006). In this study, we demonstrate that an increased expression of ADA through neutrophil incubation in plasma markedly elevate the chemotactic peptide-induced generation of oxygen radicals. Plasma contains both inhibitors and activators of inflammatory reactions e.g. fibrinogen, albumin, IgG, adenine nucleotides and components of the complement, coagulation, kallikrein-kinin and fribinolysis systems (Cronstein, 1994). We used a gentle separation procedure that does not exclude platelets but minimize the risk for cellular activation. We have unpublished results showing no differences regarding the effect of platelet free plasma and plasma with platelets on neutrophil ROS production, which indicates no involvement of platelets. In the present study, we found a high expression of ADA in plasma. ADA catalyzes the hydrolytic deamination of adenosine to inosine and thereby lowers the concentration of adenosine and counteracts the inhibitory effects of this nucleoside. Our results show that the prolonged ROS production from neutrophils incubated in plasma was completely abolished by an ADA-inhibitor. In addition, the amplified neutrophil ROS production in plasma was not further enhanced by adding ADA, while exogenous ADA stimulated the ROS production in the neutrophil control. This supports that ADA expressed in plasma stimulates neutrophil ROS production induced by fMLP. Several studies have suggested an important regulatory function of ADA in plasma and in blood cells during different infectious and cardiovascular diseases (Chavan et al., 2007, Klockars et al., 1991). At low picomolar concentrations, adenosine binds to the A1 receptor leading to potentiated neutrophil activation. Ciruela et al. demonstrated on smooth muscle cells a close interaction between ADA and the A1 receptor, which modulates binding of adenosine and the associated intracellular signalling (Ciruela et al., 1996). In the present study, we demonstrate that A1 receptor antagonists significantly inhibit the fMLP-induced CL response in neutrophils incubated with plasma. This is interesting because pharmacological manipulations of adenosine and its metabolites have predominantly affected actions mediated via A2 receptors. Very low concentration of the A1 receptor antagonists was required for an inhibitory effect. This can possibly be explained by some unspecific effects of the A1 receptor antagonists at higher concentrations. The potentiating effect of the A1 receptor agonist CPA on the fMLP-induced neutrophil ROS-response further supports a triggering effect of this receptor. On the contrary, when the neutrophils were incubated with plasma there was no
stimulatory effect of the A1 receptor agonist, which indicates that this receptor is already activated under these conditions. Taken together, these results suggest that plasma ADA lowers the concentration of adenosine which leads to adenosine A1 receptor activation and an elevated oxygen radical production in neutrophils. Furthermore, Saura et al. have shown that ADA plays a key role in the regulation of A1 receptors by accelerating ligand induced receptor desensitisation and internalisation, and that the two cell surface proteins are internalised via the same endocytic pathway (Saura et al., 1998). In support, we found that the plasma-mediated effects on neutrophil oxidase activity decreased when neutrophils were isolated from blood in the presence of ADA, which might be due to a prior down regulation of A1 receptors from the neutrophil surface. There are a number of A1 receptor agonist and antagonists that are developed for clinical application in a variety of cardiovascular disorders (Hayes, 2003, Slawsky and Givertz, 2009). Furthermore, studies have shown that adenosine A1 receptor antagonists reduce renal impairment in patients with acute heart failure (Cotter et al., 2008) and bronchoconstriction and airway inflammation in astma (Nadeem et al., 2006). It is well known that oxidative stress is an important risk factor in atherosclerosis. Consequently, the decreased neutrophil ROS production by the adenosine A1 receptor antagonists shown in this study, could explain some of the beneficial effects of the antagonists in cardiovascular disease. An elevation of cAMP, triggered by endothelial-derived adenosine or prostaglandin, is considered to turn off neutrophil activities, and thereby antagonize the harmful effects of hydrolytic enzymes and toxic oxygen radicals in the vascular space (Ottonello et al., 1995). However, during the initial phases of neutrophil activation, including transmigration through layers of platelets and endothelial cells, an elevated PDE-activity is probably needed to counteract the inhibitory effects of cAMP (Lorenowicz et al., 2007). In this study, inhibition of class IV PDEs totally abolished the plasma-mediated ROS production in neutrophils. The plasma-induced second phase of neutrophil oxidase activity was not reduced when stimulating adenylate cyclase with forskolin, which moreover confirms a high PDE activity. Furthermore, inhibition of the cAMP-dependent protein kinase A did not affect the plasma-mediated enhancement of the fMLPtriggered ROS production, indicating that the inhibitory cAMP/PKA pathway is inactive. There are by now a large amount of evidence showing that leukocytes are involved in the development of vascular diseases and that oxygen radicals have a vital role (Weber et al., 2008, Cave et al., 2006, Quinn et al., 2008). The pro-inflammatory properties of plasma, including an enhanced and prolonged ROS-release, are probably important in the defence against pathogens. However, an excessive release of oxygen radicals in the vascular space might cause persistent inflammatory processes leading to arteriosclerosis (Loscalzo, 2003). It is considered that cAMP-elevation, induced by adenosine and A2 receptor activation, is part of an endogenous mechanism which down-regulates inflammatory reactions and the progression to chronic inflammation and tissue destruction (Lorenowicz et al., 2007). This study suggests that plasma increase the activity of ADA in bacterial peptidestimulated neutrophils, and thereby counteract an adenosineinduced autoregulatory inhibitory pathway. This probably facilitates the subsequent steps in the neutrophil activation, including the emigration from the vascular compartment to the inflamed extravascular tissue. However, an overexpressed ADA-activity antagonizing the anti-inflammatory effects of adenosine may contribute to chronic inflammation. Consequently, ADA inhibition may serve as therapeutic strategy in the treatment of inflammatory disorders, such as atherosclerosis and rheumatic diseases.
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