Published online before print October 12, 2006, doi: 10.1161/01.RES.0000250174.31269.70
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2006 American Heart Association, Inc.
Cellular Biology |
ATP Release From Activated Neutrophils Occurs via Connexin 43 and Modulates Adenosine-Dependent Endothelial Cell Function
From the Department of Anesthesiology and Intensive Care Medicine (H.K.E., T.E., A.M., N.K., C.K., T.W.), Tübingen University Hospital, Germany; Institut für Pathophysiologie (K.B., R.S.), Zentrum für Innere Medizin, Universitätsklinikum Essen, Germany; Transplantation Center (S.C.R.), Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School; and Center for Experimental Therapeutics and Reperfusion Injury (S.P.C.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Holger K. Eltzschig, MD, PhD, Department of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital, Waldhörnle Str. 22, D-72072 Tübingen, Germany. E-mail heltzschig{at}partners.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Extracellular ATP liberated during hypoxia and inflammation can either signal directly on purinergic receptors or can activate adenosine receptors following phosphohydrolysis to adenosine. Given the association of polymorphonuclear leukocytes (PMNs) with adenine-nucleotide/nucleoside signaling in the inflammatory milieu, we hypothesized that PMNs are a source of extracellular ATP. Initial studies using high-performance liquid chromatography and luminometric ATP detection assays revealed that PMNs release ATP through activation-dependent pathways. In vitro models of endothelial barrier function and neutrophil/endothelial adhesion indicated that PMN-derived ATP signals through endothelial adenosine receptors, thereby promoting endothelial barrier function and attenuating PMN/endothelial adhesion. Metabolism of extracellular ATP to adenosine required PMNs, and studies addressing these metabolic steps revealed that PMN express surface ecto-apyrase (CD39). In fact, studies with PMNs derived from cd39–/– mice showed significantly increased levels of extracellular ATP and lack of ATP dissipation from their supernatants. After excluding lytic ATP release, we used pharmacological strategies to reveal a potential mechanism involved in PMN-dependent ATP release (eg, verapamil, dipyridamole, brefeldin A, 18-
-glycyrrhetinic acid, connexin-mimetic peptides). These studies showed that PMN ATP release occurs through connexin 43 (Cx43) hemichannels in a protein/phosphatase-A–dependent manner. Findings in human PMNs were confirmed in PMNs derived from induced Cx43–/– mice, whereby activated PMNs release less than 15% of ATP relative to littermate controls, whereas Cx43 heterozygote PMNs were intermediate in their capacity for ATP release (P<0.01). Taken together, our results identify a previously unappreciated role for Cx43 in activated PMN ATP release, therein contributing to the innate metabolic control of the inflammatory milieu.
Key Words: nucleotide • nucleoside • adenosine • endothelia • inflammation • ATP • connexin • inflammation • hypoxia
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Past studies have revealed a central role of extracellular nucleotide phosphohydrolysis and nucleoside signaling in innate immune responses during conditions of limited oxygen availability (hypoxia) or during acute inflammation. For example, metabolic enzymes and vascular nucleotide levels are consistently increased during hypoxia.1,2 The contribution of individual nucleotides (ATP, ADP, AMP) to these innate responses remain unclear. Polymorphonuclear granulocytes (PMNs) function as a first line of cellular response during acute inflammatory episodes.3 Previous reports have suggested that PMNs may release ATP during conditions of inflammation or hypoxia.1 Such extracellular ATP can either signal directly to vascular ATP receptors4 or may function as a metabolite following conversion via ecto-apyrase (CD39, conversion of ATP to AMP) and ecto-5'-nucleotidase (CD73, conversion of AMP to adenosine).
In the present study, we aimed to identify molecular mechanisms involved in ATP release from activated PMNs and detail consecutive metabolic and signaling pathways to modulate endothelial cell function. For this purpose, we used pharmacological and genetic approaches to inhibit ATP release from human or rodent PMNs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Isolation of Human PMNs
After approval by the Institutional Review Board and obtaining written informed consent from each individual, PMNs were freshly isolated as described previously.5,6
Preparation of Activated PMN Supernatants and Measurement of ATP or Myeloperoxidase Content
Activated PMN supernatants were prepared and ATP content measured as described previously.1
Cell Viability Assay
To evaluate lytic cell death of PMNs, lactate dehydrogenase (LDH) activity was measured in the supernatant (Roche Diagnostics).
PMN Granule Isolation
The granule fraction from PMNs was purified from resting neutrophils as previously described.7
Immunoblotting Experiments
PMNs were freshly isolated from human donors and lysed and blotted as described previously.1
Measurement of CD39 and CD73 Activity on PMNs
Surface enzyme activity was measured as described previously.1
Flow Cytometric Analysis of PMN Surface Expression of CD39 and CD73
Surface expression of CD73 and CD39 on PMNs were measured as described previously.8
Macromolecule Paracellular Permeability Assay
Endothelial permeability of cultured endothelia was measured as described previously.1
PMN Adhesion Assay
PMN adhesion was measured as described previously.5
Isolation and Activation of Murine PMNs
In subsets of experiments, PMNs were isolated from previously described mice with targeted gene deletion of cd399 or induced ablation of connexin 43 (Cx43).10 These protocols were in accordance with NIH guidelines for the use of live animals and were approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital and of the University of Essen, Germany.
Data Analysis
Data were compared by 2-factor ANOVA, or by Student’s t test where appropriate. In experiments on the kinetics of ATP release, 1 representative experiment of 3 is displayed (n=3), with triplicate measurement of the ATP concentration. Values are expressed as the mean±SD from at least 3 separate experiments.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PMNs Release ATP on Activation
We have previously shown that PMNs have the capacity to release adenine nucleotides in the form of ATP,1 although the molecular details of nucleotide release from PMNs remain largely unknown. Here, we sought to understand details of ATP release from PMNs. Initially, we determined whether ATP release was activation dependent. For these purposes, we distinguished extracellular ATP levels in the presence and absence of the potent PMN activator fMLP (N-formyl Met-Leu-Phe). In fact, ATP was readily detected in supernatants of freshly isolated PMNs (based on biophysical criteria such as retention time, coelution with internal ATP standards, and UV absorption spectra [data not shown]). In fact, ATP release increased by greater than approximately 6-fold on fMLP activation (area under curve of the high-performance liquid chromatographic [HPLC] tracing; Figure 1A). These findings from HPLC-based detection were confirmed using a luminometric ATP detection assays. As shown in Figure 1B, ATP release from freshly isolated PMNs was 4.2±1.6 nmol/107 PMNs without activation at 4°C in Ca2+-free Hank’s balanced salt solution (HBSS). Higher ATP levels were observed at 37°C in Ca2+-containing buffer (maximal levels 13.2±6.3 nmol/107 PMNs; P<0.01 by ANOVA). With fMLP activation, extracellular ATP concentrations profoundly increased, with a rapid ATP peak as early as 1 minute after fMLP activation (89±7.7 nmol/107 PMNs; P<0.001 by ANOVA), and progressively dissipated to control levels within 15 minutes. As shown in Figure 1C, repeated stimulation with fMLP results in an attenuated ATP response, with almost no stimulated ATP release on a third fMLP stimulation (Figure 1C). These results indicate a metabolic and activation-dependent release of ATP from human PMNs.
|
Mechanism of Extracellular ATP Metabolism
In the course of these studies, we addressed the rapid loss of extracellular ATP following PMN activation. In our experimental setting of 107 PMN/mL, extracellular ATP concentrations were as high as 100 nmol/L, whereas cytoplasmic concentrations were as high as 5 mmol/L (see later), thereby resulting in a 50 000-fold transmembrane ATP gradient, making passive ATP reuptake highly unlikely. As a second possibility, we considered extracellular ATP phosphohydrolysis by PMNs. A primary source of extracellular ATP phosphohydrolysis is cell surface CD39, and, therefore, we determined whether PMNs express surface CD39. For these purposes, we used a nonnative exogenous substrate (etheno-ATP), which could be distinguished from endogenous ATP via HPLC analysis. To measure CD39 activity, we quantified etheno-ATP conversion to etheno-AMP by intact PMNs (107/mL) in the presence and absence of the CD73 inhibitor -ß-methylene-ADP (APCP) (10 µmol/L). Previous studies have indicated that etheno-ATP (and etheno-AMP for CD73) can be used for measuring CD39/73 activity as they show similar conversion rates as their native compounds.1 As shown in Figure 2A, isolated PMNs rapidly metabolized etheno-ATP to etheno-AMP, suggesting high levels of CD39-activity. Surprisingly, etheno-AMP was stable in the supernatant independent of the presence of APCP (10 µmol/L), suggesting that PMNs express little or no CD73. To confirm this hypothesis, we measured CD73 activity on PMNs (conversion of etheno-AMP to etheno-adenosine).1 This analysis confirmed our inhibitor experiments and revealed no detectable CD73 on intact PMNs (Figure 2B). To confirm these results, we used fluorescence-activated cell sorting (FACS) analysis for CD39 and CD73 on various leukocyte populations. As shown in Figure 2C, PMNs and monocytes express high levels of CD39, whereas lymphocytes lack CD39 surface expression. By contrast, PMNs and monocytes express no detectable CD73, whereas CD73 is highly expressed on lymphocytes. These experiments in PMNs were repeated following fMLP stimulation and did not influence the pattern of CD39 and CD73 expression (data not shown).
|
Different Kinetics of ATP Levels Within the Supernatant of Activated PMNs Derived From cd39–/– Mice
We next extended the above findings with human PMNs to murine PMNs. For these purposes, we compared CD39 activity on isolated PMNs from cd39-null mice9 and littermate controls. Whereas PMNs from littermate controls readily converted etheno-ATP to etheno-AMP, such activity was completely absent on PMNs from cd39–/– mice (date not shown). We then measured ATP concentration in the supernatant of activated PMNs from cd39–/– mice and compared them to littermate controls. Because murine PMNs express little or no surface fMLP receptors, we used leukotriene B4 (LTB4) (100 nmol/L) for activation of PMNs.11 As shown in Figure 2D, freshly isolated PMNs from wild-type mice released ATP in an activation-dependent manner (maximal 6.7±0.57-fold increase), with similar kinetics as human PMNs (see Figure 1B). Similar to wild-type mice, PMNs from cd39–/– mice also released ATP in an activation-dependent manner. Moreover, the lack of extracellular metabolism through surface CD39 resulted in accumulation of ATP (1.6±0.09-fold increase in maximal ATP levels compared with PMNs from wild-type mice; P<0.05). Similarly, ATP levels in the supernatant of nonactivated PMNs from cd39–/– mice were higher and stayed close to their peak concentration throughout the experiment compared with wild-type PMNs (Figure 2D; P<0.05 by ANOVA). Moreover, reconstitution with 0.1 U of apyrase of supernatant from cd39–/– PMNs resulted in restoration of ATP metabolism (Figure 2E; P<0.01 by ANOVA). In addition, measurement of LDH release into the supernatant from cd39–/– or littermate controls was not different, suggesting that there is no difference in lytic ATP release (Table 1).
|
Mechanisms of PMN ATP Release
ATP exists in the cytoplasm at millimolar concentrations12 and can be released extracellularly by several mechanisms, including exocytosis of ATP-containing vesicles, transport via connexin hemichannels, through nucleoside transporters, direct transport through ATP-binding cassette (ABC) proteins or lytic cell death.12 To rule out lytic cell death as mechanism for activation-dependent ATP release, we measured LDH activity in the supernatant from activated and nonactivated PMNs. As shown in Table 1, no difference in LDH release between activated and nonactivated PMNs was found. In addition, LDH release in the supernatant has a distinctively different kinetic (nonbiphasic), suggesting that other mechanisms than lytic cell death are responsible for PMN-dependent ATP release. Next, we considered exocytosis of ATP-containing granular vesicles as a possible mechanism. To inhibit vesicular secretion, we used the general secretion inhibitor brefeldin A (BFA).13 As shown in Figure 3A, BFA (5 µg/mL) influenced neither the kinetics nor the absolute amount of ATP liberated from activated human PMNs. BFA significantly inhibited the activated release of the granule-bound enzyme myeloperoxidase (MPO) (Figure 2B). Consistent with these findings, isolated granules from resting PMNs contained greater than 95% of MPO activity (data not shown), but nearly undetectable levels of ATP, whereas cytosolic ATP concentrations were higher than 5 mmol/L (Figure 3C). Taking together these studies suggest that activation-dependent ATP release by neutrophils does not involve granular exocytosis.
|
Role of Cx43 in ATP Release From PMNs
In view of the above results that ATP is not granule bound in PMNs, we attempted pharmacological approaches to define mechanisms of ATP release. Based on reports suggesting a role of nucleoside transporter function in cellular ATP release, we examined the influence of nucleoside transport inhibitor dipyridamole (1, 10, and 100 µmol/L) on PMN ATP release.14 Dipyridamole had no effect on stimulated ATP release (Table 2). Similarly, verapamil, an inhibitor of several ABC proteins and the multidrug-resistance gene product, had no influence on ATP release. As shown in Table 2, no difference in stimulated ATP release was detectable between controls and samples treated with 1, 10, or 100 µmol/L verapamil.
|
Based on previous reports suggesting that connexin hemichannels may serve as ATP release channels in glial cells15 and the observation that PMNs express surface connexins,16 we measured ATP release of PMNs in the presence of the nonspecific gap junction inhibitor 18--glycyrrhetinic acid (18GA). As shown in Table 2, addition of 18GA resulted in a concentration-dependent inhibition of ATP release from fMLP-activated PMNs (P<0.01 by ANOVA). Additional experiments with the nonspecific gap junction inhibitor anandamide14 confirmed the above results, revealing a 4.6±0.62-fold decrease in stimulated ATP release in the presence of 100 µmol/L anandamide (P<0.01 by ANOVA; data not shown).
We extended these findings to define specific connexin contributions to PMN ATP release. For these purposes, we next used connexin mimetic peptides specifically directed against Cx40 and Cx43.16–18 As shown in Table 2, peptides specific for Cx40 did not significantly influence ATP liberation from activated PMNs. By contrast, the peptides which block Cx43 showed a concentration-dependent inhibition of ATP liberation (Table 2; P<0.01 by ANOVA), with a >6-fold reduction of maximal ATP release at 1 minute after fMLP stimulation. These results significantly implicate Cx43 in activated ATP release from human PMNs.
Activation-Dependent PMN Cx43 Dephosphorylation
It is known that hexameric assemblies of connexin 43 molecules (so called connexons) form hemichannels connecting the intracellular to the extracellular space.19 The conductance and permeability of such Cx43 hemichannels is regulated by modification of their cytoplasm domain, with phosphorylation of Ser368 causing a conformational change resulting in decreased connexon permeability.20 Therefore, we examined the influence of fMLP on Cx43 Ser368 phosphorylation in intact PMNs. As shown in Figure 4A, Cx43 is prominently phosphorylated in resting PMNs (Figure 4A, 0 minutes). Within 1 minute following fMLP activation, phosphorylation of Cx43 precipitously decreases and slowly recovers over 15 minutes. These results are consistent with fMLP-dependent dephosphorylation of Cx43 and subsequent conformational opening of Cx43 hemichannels.
|
Previous reports have implicated protein phosphatase 2A in Cx43 dephosphorylation.21 Therefore, we performed the above experiment in the presence of the protein phosphatase inhibitor okadaic acid (OA) (100 nmol/L). As show in Figure 4B, fMLP-induced dephosphorylation of Cx43 was attenuated in the presence of 100 nmol/L OA. Based on this observation, we assessed ATP release of PMNs in the presence of OA. As shown in Figure 4C, ATP of PMNs was decreased 4.1±0.3-fold in the presence of 100 nmol/L OA. Taken together, these results reveal activation-dependent dephosphorylation of Cx43 via protein phosphatase and resultant activation of ATP release in human PMNs.
Biologically Active Adenosine Liberated via PMN CD39 and Endothelial CD73
Based on the above observation that PMNs express CD39 but not CD73 on their surface, and that ATP in the presence of PMNs is rapidly hydrolyzed to AMP, we hypothesized that an additional cell type is necessary to contribute CD73-dependent AMP conversion and establish an adenosine-dependent signaling pathway.5 Because of the close spatial relationship of PMNs to the endothelium during transendothelial migration, its pivotal role to orchestrate PMN invasion into the underlying tissues during inflammatory hypoxia,4 and the fact that CD73 is induced by hypoxia on the endothelial surface,1 we examined effects of supernatants from activated PMNs on normoxic or posthypoxic endothelial cell function as a model for neutrophil/endothelial crosstalk. To pursue these experiments, we activated PMNs with fMLP and exposed HMEC-1 cells to different concentrations of the supernatant and measured paracellular barrier function, using a previously described in vitro model.1 Consistent with previous findings, endothelial exposure to the supernatant of PMNs resulted in a concentration-dependent decrease in paracellular permeability (P<0.01 by ANOVA; Figure 5A).1 Such changes in paracellular permeability were inhibited by the nonspecific adenosine receptor antagonist 8-phenyl-theophylline (10 µmol/L), thereby significantly implicating adenosine in this response. The increased barrier responses of posthypoxic endothelia are most likely attributable to hypoxia induction of CD39, CD73, and the adenosine A2B receptor, resulting in increased adenosine generation and signaling in posthypoxic endothelia.1
|
Role of Cx43-Dependent ATP Release by PMNs in Modulating Endothelial Cell Function
To investigate the role of Cx43-dependent ATP release, we next generated supernatants from fMLP-activated PMNs that were preincubated (10 minutes) and activated in the presence of 18GA (100 µmol/L) and connexin mimetic peptides (100 µmol/L). Although 18GA or connexin mimetic peptides alone did not result in a change of endothelial flux rates (data not shown), the barrier protective effects of the supernatant was absent if PMNs were activated in the presence of 18GA (10 µmol/L) or the connexin mimetic peptide specific for Cx43 (100 µmol/L). This suggests that connexin-dependent ATP release is required for the observed barrier effects of the supernatant (Figure 5B). Taken together, these results suggest that the known barrier protective effects of supernatants from activated PMNs require Cx43-dependent ATP liberation from PMNs. Moreover, these experiments also highlight the role of PMNs and endothelia as crosstalk partners in an adenosine dependent signaling pathway, with PMNs liberating ATP and CD39-dependent phosphohydrolysis to AMP, whereas endothelial CD73 activity results in the generation of adenosine and activation of endothelial adenosine receptors.
As a second model of crosstalk between PMNs and endothelia, we investigated the role of ATP release from PMNs for neutrophil adhesion to normoxic or posthypoxic endothelia. Consistent with previous studies, adhesion of fMLP-activated PMNs was increased in posthypoxic endothelia and the addition of the nonspecific adenosine receptor antagonist 8-phenyltheopylline (8-PT) (10 µmol/L)further increased PMN adhesion, suggesting that such increases in PMNs to endothelia are dependent on adenosine signaling (Figure 5C).5 As a next step, we measured PMN adhesion to normo- or posthypoxic endothelia in the presence of the connexin mimetic peptide specific for Cx40 (Figure 5C) and for Cx43 (Figure 5D). Similar to using different concentrations of the peptides alone (data not shown), the addition of the Cx40-specific peptide did not alter PMN adhesion to a measurable degree. In contrast, inhibition of ATP release from fMLP-activated PMNs with the Cx43 specific connexin mimetic peptide resulted in a concentration-dependent increase in neutrophil/endothelial adhesion.
Activated PMNs From Mice With Induced Deletion of Cx43 Show Decreased ATP Release
As proof of principle for biologically relevant PMN Cx43 activity, we isolated PMNs from age- and sex-matched mice with induced deletion of Cx43 (Cx43Cre-ER(T)/fl+4-OHT, further referred to as Cx43–/–) and the corresponding floxed control animals (Cx43fl/fl), as well as heterozygote Cx43-null mice (Cx43+/–). As depicted from Western blot analysis from cardiac tissue in Figure 6A and 6B, administration of tamoxifen resulted in nearly complete deletion of Cx43 in the Cx43–/– mice and a corresponding 50% decrease in Cx43+/– mice. Floxed control animals (Cx43fl/fl)had similar cardiac Cx43 expression to that of wild-type animals.
|
Consistent with our results from pharmacological inhibition of Cx43, isolated PMN ATP release on activation was almost completely abolished in Cx43–/– mice (P<0.001 by ANOVA compared with wild-type mice and compared with floxed controls, Figure 6C). By contrast, Cx43+/– mice had higher ATP levels than Cx43–/– knockout mice but lower than wild-type animals or floxed controls (P<0.05 compared with wild-type, floxed controls, or Cx43–/– by ANOVA). The floxed control mice had similar ATP levels than wild-type controls. As shown in Figure 6D, the total amount of PMN ATP release was closely correlated with the degree of Cx43 expression (Figure 6B). In addition, measurement of LDH release into the supernatant from Cx43–/– or Cx43fl/fl-control mice was not different, suggesting that there is no difference in lytic ATP release (Table 1). These studies provide genetic evidence that ATP release from activated PMNs occurs in a Cx43-dependent fashion.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Metabolic and transcriptional responses to inflammation are common denominators of multiple cardiovascular and pulmonary diseases. In particular, adaptation to "inflammatory hypoxia" has become an area of intense investigation.4 Important in this regard, a consistent finding in hypoxic tissues is increased extracellular nucleotide levels.1 Here, we identified a novel role for Cx43 in activation-dependent ATP release from PMNs. Moreover, ATP is rapidly metabolized to AMP through catalytic activity involving PMN surface CD39. Confirmatory studies in inducible cx43-deficient mice revealed that Cx43 expression correlated with PMN ATP release. Taken together, these studies demonstrate nucleotide liberation at sites of acute inflammation by PMNs and identify Cx43-dependent ATP release as a central part of an innate inflammatory response controlling adenosine-dependent endothelial function.
We observed a barrier-protective and antiinflammatory role of ATP released from PMNs resulting from rapid metabolism to adenosine. In fact, previous studies using specific adenosine receptor antagonists (eg, MRS 1754) have revealed a pivotal role of adenosine receptors (particularly A2A and A2B) on PMNs in modulating neutrophil/endothelial crosstalk pathways during conditions of limited oxygen availability.5 Consistent with our findings, previous studies indicated that Cx43 phosphorylation can be modulated by inflammation and hypoxia, resulting in an alteration in cellular functions. For instance, dephosphorylation of Cx43 and uncoupling of myocardial gap junctions occurs during myocardial ischemia. Under such circumstances, Cx43 may be reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation dependent on fluctuations in intracellular ATP content.22 Moreover, several studies have implicated a role for Cx43 in cardioprotection by ischemic preconditioning, insomuch as protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous Cx43-deficient mice.23 In view of the results from the present study showing a critical role of Cx43 as a phosphorylation-dependent ATP release channel on PMNs in modulating endothelial adenosine responses, it is tempting to speculate that the role of Cx43 as ATP channel may also be involved in cardioprotection by ischemic preconditioning. In fact, this may point to a clinical role of Cx43-dependent ATP release in myocardial ischemia. Previous studies have demonstrated extracellular adenosine generation by the ecto-5'-nucleotidase (CD73) in cardiac ischemic preconditioning.24 However, the source from which extracellular nucleotide precursor molecules are generated and which cells contribute to their release remains unknown. Thus, PMN-dependent ATP release could represent an important substrate for nucleotidase-dependent extracellular adenosine generation during cardioprotection by ischemic preconditioning. However, pharmacological strategies to modulate Cx43-dependent ATP release and studies using a chimeric approach or tissue-specific gene deletion of Cx43 will have to confirm a role of Cx43-dependent ATP release from PMNs in acute myocardial ischemia.
In summary, our results highlight for the first time a critical role of Cx43 on the surface of PMNs in releasing ATP from inflammatory cells during activation. Such ATP is rapidly hydrolyzed to adenosine via close association with CD73 expressing cell types. Thus, PMN-dependent release of ATP may play a critical role in the metabolic control of innate inflammatory pathways.
|
Acknowledgments |
|---|
We acknowledge Stephanie Zug, Marion Faigle, and Edgar Hoffmann for technical assistance and Shelley K. Eltzschig for the artwork. We acknowledge Dr R. John MacLeod for valuable advice at the initial stages of this work.
Sources of Funding
This work was supported by Fortune grant 1416-0-0 and Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Verbundprojekt grant 1597-0-0 from the University of Tübingen (to H.K.E.); Deutsche Forschungsgemeinschaft (DFG) grant EL274/2-2 (to H.K.E.); IZKF Nachwuchsgruppe of the University of Tübingen (grant 1605-0-0 to T.E.); and NIH grants HL60569 and DK50189 (to S.P.C.).
Disclosures
None.
|
Footnotes |
|---|
Original received July 8, 2006; resubmission received August 28, 2006; accepted October 3, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Eltzschig HK, Ibla JC, Furuta GT, Leonard MO, Jacobson KA, Enjyoji K, Robson SC, Colgan SP. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J Exp Med. 2003; 198: 783–796.
2. Thompson LF, Eltzschig HK, Ibla JC, Van De Wiele CJ, Resta R, Morote-Garcia JC, Colgan SP. Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia. J Exp Med. 2004; 200: 1395–1405.
3. Cramer T, Yamanishi Y, Clausen BE, Forster I, Pawlinski R, Mackman N, Haase VH, Jaenisch R, Corr M, Nizet V, Firestein GS, Gerber HP, Ferrara N Johnson RS. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell. 2003; 112: 645–657.[CrossRef][Medline] [Order article via Infotrieve]
4. Weissmuller T, Eltzschig HK, Colgan SP. Dynamic purine signaling and metabolism during neutrophil-endothelial interactions. Purinergic Signal. 2005; 1: 229–239.[Medline] [Order article via Infotrieve]
5. Eltzschig HK, Thompson LF, Karhausen J, Cotta RJ, Ibla JC, Robson SC, Colgan SP. Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism. Blood. 2004; 104: 3986–3992.
6. Eltzschig HK, Weissmuller T, Mager A, Eckle T. Nucleotide metabolism and cell-cell interactions. Methods Mol Biol. 2006; 341: 73–87.[Medline] [Order article via Infotrieve]
7. Kjeldsen L, Sengelov H, Borregaard N. Subcellular fractionation of human neutrophils on Percoll density gradients. J Immunol Methods. 1999; 232: 131–143.[CrossRef][Medline] [Order article via Infotrieve]
8. Kong T, Eltzschig HK, Karhausen J, Colgan SP, Shelley CS. Leukocyte adhesion during hypoxia is mediated by HIF-1-dependent induction of {beta}2 integrin gene expression. Proc Natl Acad Sci U S A. 2004; 101: 10440–10445.
9. Enjyoji K, Sevigny J, Lin Y, Frenette PS, Christie PD, Esch JS 2nd, Imai M, Edelberg JM, Rayburn H, Lech M, Beeler DL, Csizmadia E, Wagner DD, Robson SC, Rosenberg RD. Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med. 1999; 5: 1010–1017.[CrossRef][Medline] [Order article via Infotrieve]
10. Eckardt D, Theis M, Degen J, Ott T, van Rijen HV, Kirchhoff S, Kim JS, de Bakker JM, Willecke K. Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion. J Mol Cell Cardiol. 2004; 36: 101–110.[CrossRef][Medline] [Order article via Infotrieve]
11. Haribabu B, Verghese MW, Steeber DA, Sellars DD, Bock CB, Snyderman R. Targeted disruption of the leukotriene B4 receptor in mice reveals its role in inflammation and platelet-activating factor-induced anaphylaxis. J Exp Med. 2000; 192: 433–438.
12. Novak I. ATP as a signaling molecule: the exocrine focus. News Physiol Sci. 2003; 18: 12–17.
13. Maroto R, Hamill OP. Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction. J Biol Chem. 2001; 276: 23867–23872.
14. Wang ECY, Lee JM, Ruiz WG, Balestreire EM, von Bodungen M, Barrick S, Cockayne DA, Birder LA, Apodaca G. ATP and purinergic receptor-dependent membrane traffic in bladder umbrella cells. J Clin Invest. 2005; 115: 2412–2422.[CrossRef][Medline] [Order article via Infotrieve]
15. Stout CE, Costantin JL, Naus CCG, Charles AC. Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem. 2002; 277: 10482–10488.
16. Oviedo-Orta E, Evans WH. Gap junctions and connexins: potential contributors to the immunological synapse. J Leukoc Biol. 2002; 72: 636–642.
17. Leybaer L, Braet K, Vandamme W, Cabooter L, Martin PE, Evans WH. Connexin channels, connexin mimetic peptides and ATP release. Cell Commun Adhes. 2003; 10: 251–257.[Medline] [Order article via Infotrieve]
18. Oviedo-Orta E, Errington RJ, Evans WH. Gap junction intercellular communication during lymphocyte transendothelial migration. Cell Biol Int. 2002; 26: 253–263.[CrossRef][Medline] [Order article via Infotrieve]
19. Goodenough DA, Paul DL. Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol. 2003; 4: 285–294.[CrossRef][Medline] [Order article via Infotrieve]
20. Bao X, Reuss L, Altenberg GA. Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of serine 368. J Biol Chem. 2004; 279: 20058–20066.
21. Ai X, Pogwizd SM. Connexin 43 downregulation and dephosphorylation in nonischemic heart failure is associated with enhanced colocalized protein phosphatase type 2A. Circ Res. 2005; 96: 54–63.
22. Turner MS, Haywood GA, Andreka P, You L, Martin PE, Evans WH, Webster KA, Bishopric NH. Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels. Circ Res. 2004; 95: 726–733.
23. Schwanke U, Konietzka I, Duschin A, Li X, Schulz R, Heusch G. No ischemic preconditioning in heterozygous connexin43-deficient mice. Am J Physiol Heart Circ Physiol. 2002; 283: H1740–H1742.[Medline] [Order article via Infotrieve]
24. Kitakaze M, Hori M, Morioka T, Minamino T, Takashima S, Sato H, Shinozaki Y, Chujo M, Mori H, Inoue M. Infarct size-limiting effect of ischemic preconditioning is blunted by inhibition of 5'-nucleotidase activity and attenuation of adenosine release. Circulation. 1994; 89: 1237–1246.
This article has been cited by other articles:
 |
|
 |
|
  T. Eckle, M. Koeppen, and H. K. Eltzschig Role of Extracellular Adenosine in Acute Lung Injury Physiology, October 1, 2009; 24(5): 298 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hatfield, B. Belikoff, D. Lukashev, M. Sitkovsky, and A. Ohta The antihypoxia-adenosinergic pathogenesis as a result of collateral damage by overactive immune cells J. Leukoc. Biol., September 1, 2009; 86(3): 545 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Reutershan, I. Vollmer, S. Stark, R. Wagner, K.-C. Ngamsri, and H. K. Eltzschig Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs FASEB J, February 1, 2009; 23(2): 473 - 482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Corriden, Y. Chen, Y. Inoue, G. Beldi, S. C. Robson, P. A. Insel, and W. G. Junger Ecto-nucleoside Triphosphate Diphosphohydrolase 1 (E-NTPDase1/CD39) Regulates Neutrophil Chemotaxis by Hydrolyzing Released ATP to Adenosine J. Biol. Chem., October 17, 2008; 283(42): 28480 - 28486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Eckle, L. Fullbier, A. Grenz, and H. K. Eltzschig Usefulness of pressure-controlled ventilation at high inspiratory pressures to induce acute lung injury in mice Am J Physiol Lung Cell Mol Physiol, October 1, 2008; 295(4): L718 - L724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. P. Veliz, F. G. Gonzalez, B. R. Duling, J. C. Saez, and M. P. Boric Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1056 - H1066. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Hart, M. Henn, D. Kohler, D. Kloor, M. Mittelbronn, I. C. Gorzolla, G. L. Stahl, and H. K. Eltzschig Role of extracellular nucleotide phosphohydrolysis in intestinal ischemia-reperfusion injury FASEB J, August 1, 2008; 22(8): 2784 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Hart, D. Kohler, T. Eckle, D. Kloor, G. L. Stahl, and H. K. Eltzschig Direct Treatment of Mouse or Human Blood With Soluble 5'-Nucleotidase Inhibits Platelet Aggregation Arterioscler Thromb Vasc Biol, August 1, 2008; 28(8): 1477 - 1483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Blum, S. M. Joseph, R. J. Przybylski, and G. R. Dubyak Rho-family GTPases modulate Ca2+-dependent ATP release from astrocytes Am J Physiol Cell Physiol, July 1, 2008; 295(1): C231 - C241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Esther Jr, N. E. Alexis, M. L. Clas, E. R. Lazarowski, S. H. Donaldson, C. M. Pedrosa Ribeiro, C. G. Moore, S. D. Davis, and R. C. Boucher Extracellular purines are biomarkers of neutrophilic airway inflammation Eur. Respir. J., May 1, 2008; 31(5): 949 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Eckle, M. Faigle, A. Grenz, S. Laucher, L. F. Thompson, and H. K. Eltzschig A2B adenosine receptor dampens hypoxia-induced vascular leak Blood, February 15, 2008; 111(4): 2024 - 2035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Auchampach Adenosine Receptors and Angiogenesis Circ. Res., November 26, 2007; 101(11): 1075 - 1077. [Full Text] [PDF]
|
|
|
|
 |
|
 D. Kohler, T. Eckle, M. Faigle, A. Grenz, M. Mittelbronn, S. Laucher, M. L. Hart, S. C. Robson, C. E. Muller, and H. K. Eltzschig CD39/Ectonucleoside Triphosphate Diphosphohydrolase 1 Provides Myocardial Protection During Cardiac Ischemia/Reperfusion Injury Circulation, October 16, 2007; 116(16): 1784 - 1794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Corriden, P. A. Insel, and W. G. Junger A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1420 - C1425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Grenz, H. Zhang, M. Hermes, T. Eckle, K. Klingel, D. Y. Huang, C. E. Muller, S. C. Robson, H. Osswald, and H. K. Eltzschig Contribution of E-NTPDase1 (CD39) to renal protection from ischemia-reperfusion injury FASEB J, September 1, 2007; 21(11): 2863 - 2873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang, M. Ma, S. Locovei, R. W. Keane, and G. Dahl Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters Am J Physiol Cell Physiol, September 1, 2007; 293(3): C1112 - C1119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Loffler, J. C. Morote-Garcia, S. A. Eltzschig, I. R. Coe, and H. K. Eltzschig Physiological Roles of Vascular Nucleoside Transporters Arterioscler Thromb Vasc Biol, May 1, 2007; 27(5): 1004 - 1013. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Eckle, T. Krahn, A. Grenz, D. Kohler, M. Mittelbronn, C. Ledent, M. A. Jacobson, H. Osswald, L. F. Thompson, K. Unertl, et al. Cardioprotection by Ecto-5'-Nucleotidase (CD73) and A2B Adenosine Receptors Circulation, March 27, 2007; 115(12): 1581 - 1590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Grenz, H. Zhang, T. Eckle, M. Mittelbronn, M. Wehrmann, C. Kohle, D. Kloor, L. F. Thompson, H. Osswald, and H. K. Eltzschig Protective Role of Ecto-5'-Nucleotidase (CD73) in Renal Ischemia J. Am. Soc. Nephrol., March 1, 2007; 18(3): 833 - 845. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||