Adenosine Inhibits Matrix Metalloproteinase-9 Secretion By Neutrophils
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Isabelle Ernens, Didier Rouy, Emilie Vel
参见附件。
the Laboratory of Cardiovascular Research (I.E., D.R., E.V., Y.D.), CRP-Santé, Luxembourg
the Division of Cardiology (D.R.W.), Centre Hospitalier Luxembourg.
Abstract
Matrix metalloproteinases (MMPs), and in particular MMP-9 secreted by neutrophils, are capable of degrading the matrix components of the heart and are thought to be the driving force behind myocardial matrix remodeling after infarction. Adenosine, a naturally produced nucleoside, has been shown to have cardioprotective effects and to inhibit secretion of various cytokines. The aim of our study was to determine the effect of adenosine on the secretion of MMP-9 by neutrophils. Neutrophils were isolated from healthy volunteers through Ficoll and Dextran sedimentation. Neutrophils were activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence or absence of adenosine or adenosine analogs. Zymography and enzyme linked immunosorbent assay were used to measure MMP-9 secretion. Adenosine (1 μmol/L) decreased the fMLP-induced MMP-9 secretion by 30±2% (n=8, P<0.001). The effect was dose-dependent and was not specific to fMLP because adenosine also inhibited MMP-9 secretion by LPS- or H2O2-stimulated neutrophils. The effect of adenosine was mimicked by the adenosine A2a receptor agonist CGS21680 and was inhibited by both the A2a antagonist SCH5826 and A2a RNA silencing. The A3 agonist IB-MECA moderately decreased fMLP-induced MMP-9 secretion. Agonists and antagonists of the other types of adenosine receptors had no significant effect. Adenosine increased intracellular cAMP concentration and accelerated the return to baseline of the intracytoplasmic calcium peak. The inhibition of MMP-9 secretion by adenosine, as well as the calcium effect, was prevented by the protein kinase A inhibitor H-89. In conclusion, we show here that adenosine inhibits MMP-9 secretion by neutrophils. Our results suggest that this effect implies the A2a receptor and is mediated through the cAMP/PKA/Ca2+ pathway. Therefore, adenosine may represent a new approach to prevent matrix degradation and remodeling after myocardial injury.
Key Words: adenosine neutrophils myocardial remodeling protein kinase A matrix metalloproteinase-9
Introduction
Despite reperfusion therapy with thrombolysis or percutaneous intervention, the development of left ventricular remodeling and heart failure after myocardial infarction has not significantly decreased and remains associated with a more than 10-fold elevated risk of death.1 Enhanced activity of matrix metalloproteinases (MMPs) and in particular MMP-9 after infarction breaks down collagen contributing to myocyte slippage and ventricular remodeling. Recent observations2 and the Framingham study have shown that levels of MMP-9 are elevated after myocardial infarction both in the acute and chronic phases.3 We have identified MMP-9 as a risk marker for the development of left ventricular remodeling and heart failure after acute myocardial infarction.4
Animal studies have shown that ventricular remodeling after infarction can be attenuated through the use of MMPs inhibitors.5–7 However, recent results from the PREMIER (Prevention of Myocardial Infarction Early Remodeling) trial failed to demonstrate a beneficial effect of MMP inhibition on left ventricular remodeling after myocardial infarction.8 Therefore, understanding the cellular and molecular triggers of MMPs expression is of major interest to understand and possibly modify the remodeling process.
The nucleoside adenosine (Ado) is formed in the myocardium through dephosphorylation of AMP when there is an imbalance between tissue oxygen supply and demand which occurs in the setting of acute myocardial infarction and congestive heart failure. Multiple lines of evidence have shown that Ado protects against ischemia/reperfusion injury.9 In animals and in humans, Ado can reduce infarct size and improve coronary flow.10 We have shown that Ado inhibits the expression of TNF- in the rat and failing human heart.11,12 However, the effect of Ado on enzymes participating in extracellular matrix degradation has not been studied.
As high levels of MMP-9 are associated with a deleterious outcome after myocardial infarction, the aim of the present study was to investigate the effect of Ado on the expression and secretion of MMP-9. Using near-infrared fluorescent imaging, Chen et al have elegantly demonstrated that infiltrating neutrophils are the likely source of MMP-9 activity early after infarction.13 Therefore, we used isolated neutrophils to study the effect of Ado and Ado analogs on MMP-9. Taken together, our results suggest that pharmacological inhibition of MMP-9 through Ado may favor the stabilization of the extracellular matrix after myocardial infarction.
Materials and Methods
An expanded Materials and Methods section can be found in the supplement available at http://circres.ahajournals.org.
Cell Culture
Neutrophils from healthy donors were isolated from heparinized blood by Ficoll-Hypaque (ICN) centrifugation followed by dextran sedimentation (Amersham, Rosendaal Netherland). Purity of the cell preparations was >93% as determined with a BD FACSCanto flow cytometer (Beckton Dickinson, Erembodegem, Belgium) using anti-CD16 for neutrophils (Immunotools, Friesoythe, Germany). Cell viability, as determined by trypan blue exclusion, was >98%. Purified neutrophils were resuspended at a density of 0.5x106 cells/mL in RPMI 1640 medium containing L-glutamine (Cambrex, Verviers, Belgium) and supplemented with nonessential amino acids and sodium pyruvate. One ml of the cell suspension was seeded per well into 24-well plates. Cells were allowed to rest for 2 hours at 37°C-5% CO2. If indicated, neutrophils were primed with TNF- (10 U/mL) for 15 minutes before preincubation. Cells were preincubated for 5 minutes with Ado (0.01 to 25 μmol/L), Ado receptors agonists (0.1 to 100 nmol/L), or Ado receptors antagonists (1 to 100 nmol/L) as needed. Then fMLP (10–7mol/L), LPS (10 ng/mL), or H2O2 (10 mmol/L) were added and cells were incubated for another 15 minutes (H2O2 and fMLP), or 2 hours for conditions with LPS. For experiments involving siRNAs, neutrophils were transfected using the Nucleofection technology (Amaxa, Cologne, Germany): 1.4 μg of each siRNA was transfected into 1x106 cells following the manufacturer’s recommendations. Controls underwent the same transfection procedure. Cells were incubated for 48 hours before treatments with Ado and fMLP.
Monocytes were purified from peripheral blood mononuclear cells (PBMCs) obtained by Ficoll gradient using the Monocyte Isolation Kit II from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). Purity was estimated by flow cytometry with anti–CD14-PE (monocytes/macrophages), anti–CD13-APC (monocytes and granulocytes), anti–CD20-PE Cy7 (B cells), anti–CD27-FITC (T cells), CD34-PerCP (bone marrow-derived progenitor cells), and anti–CD45-APC Cy7 (leukocytes). Purity was typically above 93%. Monocytes were cultured in RPMI 1640 containing L-Glutamine, sodium pyruvate, nonessential amino acids, penicillin/streptomycin, and 1% fetal calf serum. Differentiation was achieved with 50 ng/mL macrophage-colony stimulating factor (M-CSF) for 7 days. Cells were then preincubated with 10 μmol/L Ado and 10 μmol/L erythro-9-(2-Hydroxy-3-nonyl)-adenine hydrochloride (EHNA, an adenosine deaminase inhibitor) for 15 minutes before activation with 100 ng/mL LPS for 24 hours.
For all cells, conditioned medium was collected, mixed with bovine serum albumin (0.2% final concentration) and protease inhibitors (Roche, Mannheim, Germany), and centrifuged at 600g for 5 minutes, 4°C. Cell-free supernatants were stored at –20°C until use.
Analysis of Gelatinase Activity
Gelatin zymography was performed on cell-free conditioned medium as described before.14 Densitometry was performed on scanned zymograms using the Scion Image program (Scion Corp, Frederick, Mass). The area of the pro–MMP-9 band was used to evaluate gelatinolytic activity, expressed as a percentage of the control condition as indicated. We had previously verified that this band was representative of the global MMP-9 activity (proMMP-9, lipocalin-MMP-9 complex, MMP-9 dimers). A strong correlation was observed between the intensities of the three bands.
Measurement of cAMP Concentration
cAMP concentration was assessed using the direct cAMP enzyme immunoassay kit according to the manufacturer’s protocol. Because of low cAMP concentrations, 3x106 cells were incubated in 6-well plates and the acetylated version of the kit was used. Detection limit of the assay is 0.037 pmol/mL.
Measurement of the Intracellular Calcium Concentration
For these experiments, Fluo-4/AM was used as the fluorescent, Ca2+-sensitive indicator. Neutrophils (1x106 ml–1) were preloaded with Fluo-4/AM (1 μmol/L) and Pluronic (0.02%) at room temperature on a shaker in Hanks balanced salt solution (HBSS, pH 7.4). After a 30-minute incubation, cells were washed twice in indicator-free HBSS and then incubated in HBSS for a further 15 minutes at room temperature in presence or absence of H89 (10 μmol/L). 0.5x106 cells were transferred to 96 wells plate or BD Falcon tubes (5 mL) for analysis at 37°C using a POLARstar OPTIMA fluorescence spectrophotometer (ex 490 nm and em 520 nm) and a BD FACSCanto flow cytometer (ex 488 nm and em 530/30 nm), respectively. After a stable baseline was obtained (1 minute), the neutrophils were incubated with or without Ado (1 μmol/L) during 1.5 minutes and finally were activated by addition of fMLP (10–7M). The subsequent increase in Fluo-4/AM fluorescence intensity was monitored more than a 5 minutes period.
Cytotoxicity Assay
To determine a potential cytotoxicity of any of the drugs used in our experiments, cell supernatants were assayed for lactate dehydrogenase (LDH) activity using a LDH-based cytotoxicity detection kit (Roche) as described by the manufacturer. None of the drugs used induced a significant cytotoxicity.
Data Analysis
The results are shown as mean±SEM. Most of the experiments are the mean of 3 independent experiments, unless otherwise stated. Statistical analysis was performed after passing a normality test through the GraphPad Instat program, using one-way t test or one-way ANOVA, and P<0.05 was considered to be significant.
Results
Adenosine Inhibits MMP-9 Secretion By Neutrophils
Neutrophils were preincubated for 5 minutes with Ado (0.01 to 25 μmol/L) or medium alone before fMLP stimulation for 15 minutes. Gelatinase activity was measured in cell-free conditioned medium by zymography. As shown in Figure 1A, three forms of MMP-9 were detected: pro–MMP-9, MMP-9-lipocalin complex, and pro–MMP-9 homodimer, an observation consistent with previous publications.15 Addition of fMLP induced a 2-fold increase of MMP-9 secretion as compared with control conditions. Whereas Ado had no effect on basal MMP-9 secretion (not shown), it significantly decreased all 3 forms of MMP-9 in a concentration-dependent manner in fMLP-treated neutrophils. The maximal effect of Ado was observed at 1 μmol/L with a 30±2% (n=8, P<0.001) inhibition of pro–MMP-9 secretion (Figure 1A). All 3 forms of MMP-9 were decreased to the same extent. The results obtained by zymography were confirmed by ELISA (Figure 1B). Of note, an Ado concentration of 1 μmol/L can be observed in vivo in conditions like heart failure16 and sepsis.17
Because neutrophils are likely to be primed in the context of myocardial injury, we verified that Ado was able to inhibit MMP-9 secretion by neutrophils primed with TNF- before treatment. Priming did not affect the response of neutrophils to Ado and fMLP (results not shown).
Although 15 minutes of stimulation with fMLP is likely too short to induce a de novo synthesis of the MMP-9 protein, PCR was performed and revealed that neither fMLP nor Ado had an effect on MMP-9 mRNA expression in our experimental conditions (results not shown). This observation confirmed that the increase of MMP-9 in cell-culture supernatants is solely attributable to neutrophil degranulation. To test whether the Ado effect was limited to MMP-9 (tertiary granules), culture supernatants were assessed for MMP-8 (secondary granules) and MPO (primary granules) contents. Figure 1C clearly shows that Ado also reduces the fMLP-induced release of MMP-8 and MPO, arguing for a non granule-specific effect of Ado.
The Inhibition Is Not fMLP-Dependent, and Is Adenosine-Related
To verify whether the inhibition of MMP-9 secretion by Ado was specific to fMLP, LPS and H2O2 were used as additional stimulators of MMP-9 secretion. Neutrophils were preincubated for 5 minutes with Ado (1 μmol/L) or medium before stimulation with either LPS (10 ng/mL) or H2O2 (10 mmol/L). 1% autologous serum was added to the culture medium used for LPS experiments. Similarly to fMLP-treated cells, Ado inhibited MMP-9 secretion by neutrophils stimulated with LPS or H2O2 (Figure 2A). This observation indicated that the effect of Ado on MMP-9 secretion was not restricted to the bacterial agents fMLP or LPS but also occurs when cells are stimulated with a reactive oxygen species generator such as H2O2.
The so-called Ado modulators ITU (an inhibitor of Ado kinase) and DIP (an inhibitor of Ado transport) are substances known to increase the endogenous concentration of Ado. We tested their ability to replicate the inhibition of MMP-9 secretion by Ado. Both adenosine modulators reproduced the effect of Ado (Figure 2B). Furthermore, replacing Ado by the nonspecific Ado receptor agonist NECA resulted in a similar inhibition of MMP-9 secretion (Figure 2B). These data reinforced our finding that Ado is a reproducible inhibitor of MMP-9 secretion.
Inhibition of MMP-9 Secretion by Ado Is Mediated by the A2a Receptor
To evaluate the type of Ado receptor involved in the inhibition of MMP-9 secretion by Ado, a pharmacological approach using several Ado receptors agonists and antagonists was initially chosen. The A2a-specific agonist CGS21680 was able to replicate the inhibitory effect of Ado on MMP-9. The A3-specific agonist IB-MECA showed a weaker effect on MMP-9 at high concentration whereas the A1 specific agonist C8031 was essentially without effect (Figure 3A). Among the 4 Ado receptors antagonists tested, only the A2a-specific antagonist SCH58261 was able to suppress the effect of Ado on MMP-9 (Figure 3B). To rule out the possibility that this suppression could be a result of an increase in MMP-9 secretion by SCH58261 itself, additional experiments were performed. The A2a antagonist SCH58261 at the concentration of 100 nM used in these experiments and giving maximal inhibition of MMP-9 release did not modify the fMLP-induced MMP-9 secretion (results not shown).
To compensate for a potential lack of specificity of the Ado agonists and antagonists, a second approach taking profit of the highly specific RNA interference process was used. Neutrophils were transfected with siRNA specific for the A2aAR or A3AR receptors before treatment with Ado and fMLP. Figure 3C shows that only the A2aAR-specific siRNA reversed, at least partly, the inhibitory effect of Ado on MMP-9 secretion. Taken together, these results demonstrate that the inhibition of MMP-9 secretion by Ado is most probably mediated by the A2aAR.
Human Neutrophils Express Both A2aAR and A3AR mRNA
The relative expression of the 4 different Ado receptors on neutrophils is poorly characterized. Using quantitative PCR, we confirmed the presence of both the A3 and the A2a receptors on neutrophils. The absence of any amplified products in a control reaction without reverse transcriptase (-RT) attested the absence of any contaminating genomic DNA in the RNA preparations (Figure 4A). Quantification revealed that the A2a receptor was 2.5-fold higher expressed than the A3 receptor (Figure 4B). These data are in accordance with the results obtained with Ado receptor agonists and antagonists, ie, the A2a receptor appears to be the key mediator of the inhibition of MMP-9 secretion by Ado.
The Inhibition of MMP-9 Secretion by Ado Is cAMP-, PKA-, and Calcium-Related
The bacterial agent fMLP is known to trigger neutrophil degranulation through a massive inositol triphosphate-mediated calcium release from intracellular Ca2+ stores. We investigated whether the cAMP/PKA pathway could be responsible for the observed effect of Ado through modulation of intracellular Ca2+ stores. The effect of Ado on intracellular cAMP in unstimulated and fMLP-stimulated neutrophils is shown in Figure 5. Ado increased cAMP in unstimulated and fMLP-stimulated cells to the same extent.
Because cAMP formation leads to PKA phosphorylation, the involvement of PKA in the effect of Ado was evaluated using the specific PKA inhibitor H89. When added to untreated neutrophils or fMLP-treated neutrophils, H89 did not modify MMP-9 secretion. However, it abrogated the inhibition of MMP-9 secretion by Ado (Figure 6).
Finally, the kinetic of free cytosolic calcium concentration was studied less than fMLP stimulation, in the presence or absence of Ado. Figure 7A shows a representative pattern of Fluo-4 responses in fMLP-activated neutrophils obtained by spectrofluorimetry. The addition of fMLP to neutrophils triggered the characteristic, abrupt increase in Fluo-4 fluorescence because of elevated concentrations of cytosolic Ca2+. Ado did not modify the magnitude of this increase but accelerated the rate of subsequent decline in fluorescence intensity, indicating an accelerated clearance of Ca2+ from the cytosol. Figure 7B shows that Ado shortens the time to half-peak from 43.7±11.4 sec to 23.5±6.3 sec (P<0.001), an effect totally reversed by adding H89. These results were confirmed by flow cytometry (not shown). Moreover, a reproductive decrease of cytosolic calcium by 24±7% (P<0.001) was observed during the preincubation of the neutrophils with Ado before stimulation with fMLP, indicating that Ado has an effect per se on cytosolic calcium. Signal transduction pathways activated by fMLP in human neutrophils have been recently reviewed and include the protein kinase C (PKC) pathway.18 Using the PKC inhibitors chelerythrine and BIS10, we ruled out any involvement of the PKC pathway in the inhibition of MMP-9 secretion by Ado (results not shown).
Ado Does Not Inhibit MMP-9 Secretion by Monocytes/Macrophages
Although neutrophils constitute the first wave of inflammatory cells recruited to the infarcted area, other cell types are operative in the context of myocardial injury, such as macrophages and fibroblasts. We tested whether Ado modifies the extent of MMP-9 secretion by cells from the monocytes/macrophages lineage. The effect of Ado was studied in monocytes, LPS-activated monocytes, and M-CSF–differentiated monocytes. On stimulation, MMP-9 release was gradually increased with the degree of activation and maturation of monocytes. However, in contrast to neutrophils, Ado did not significantly modify MMP-9 release in monocytes, LPS activated monocytes, or M-CSF–differentiated monocytes (Figure 8).
Discussion
In this study, we demonstrate for the first time that Ado inhibits fMLP-induced MMP-9 secretion in human neutrophils. We also provide evidence that the mechanism of the inhibitory effect of Ado on the fMLP-induced MMP-9 secretion is mediated by the A2aAR through a cAMP-PKA pathway and acceleration of Ca2+ clearance from the cytosol of activated neutrophils.
Ado has been shown to attenuate major neutrophil functions including cell adhesion, superoxyde anion production, and phagocytosis, effects mainly dependent on activation of the A2aAR. However, the effect of Ado on neutrophil degranulation has been only partially described. Ado inhibits the secretion of lysozyme,19 myeloperoxidase,20 and elastase,21 3 enzymes stored in the primary granules. The effect of Ado on gelatinase granules or tertiary granules involved in matrix degradation and diapedesis of neutrophils22 has not been studied. Gelatinase granules contain MMP-9, a member of the MMPs family known to play a pivotal role in the degradation of type IV collagen in basement membranes during left ventricular remodeling.
Our results confirm the presence of the A2aAR on neutrophils and show that A3 receptors are also present on neutrophils. So far, the A3AR protein has only been identified in human eosinophils23 and monocytes24 and in a rat basophil leukemia cell line,25 despite the detection of A3AR gene transcripts in a variety of species and tissues. Pharmacological studies using agonists and antagonists identified A3AR in human neutrophils and promyelocytic HL-60 cells.26–29
Using both pharmacological (Ado agonists and antagonists) and molecular (siRNAs) tools, we were able to show that Ado inhibits the secretion of MMP-9 mainly through its A2aAR. A small participation of the A3AR cannot completely be excluded. It has to be kept in mind that the A3 agonist used, IB-MECA, may not be as specific as previously thought. Indeed, data from a recent report suggest that IB-MECA may also bind to the A2aAR, which may explain some of the effects we observed with that compound.30 A limitation of the experiments involving RNA interference is related to the weak transcriptional machinery of neutrophils. However, our data show that cells transfected with the A2aAR-specific siRNA reproducibly lacked the inhibitory action of Ado on MMP-9 secretion.
In general, occupancy of A2aAR stimulates accumulation of intracellular cAMP, which acts as a second messenger to alter cellular functions.27 Our results suggest that the intracellular mechanism responsible for the inhibition of MMP-9 secretion by Ado involves cAMP, PKA, and intracellular calcium stores. This is supported by the fact that cAMP concentrations increase significantly after the addition of Ado and by the observation that the specific PKA inhibitor H89 completely suppresses the effect of Ado on MMP-9. The rise in cAMP after addition of Ado has been well documented.31,32 Because adenylate cyclase is activated by Gs protein, the increase in cAMP is consistent with involvement of A2aAR, a Gs-coupled receptor.
Intracellular calcium increases after neutrophil stimulation,33 leading to degranulation. On the other hand, it is known that PKA is associated with a decrease in intracellular calcium, possibly through activation of phospholamban and/or sarcoplasmic reticulum–associated Ca-ATPase. We show here that Ado does not alter the peak calcium levels but markedly accelerated the normalization of calcium levels after stimulation. This is associated with significant inhibition of MMP-9. Our study is in accordance with recent reports showing that the A2aAR may antagonize proinflammatory stimuli and restore calcium homeostasis in neutrophils.26,34
The action of Ado highlighted in this article appears to be restricted to certain cell types participating in the process of myocardial injury and remodeling. Whereas Ado reproducibly inhibited MMP-9 secretion by neutrophils, it had no effect on other inflammatory cells such as monocytes/macrophages. This is in line with recent data from Spinale et al35 showing a reduction in left ventricular remodeling where MMP activity is inhibited early on after myocardial infarction but not at a later stage. Thus, there appears to be a time window for MMP inhibition after infarction.
In conclusion, our results suggest that, in addition to the known effects of Ado on myocardial reperfusion injury and infarct size, Ado may also have a beneficial effect at the level of the extracellular matrix. Indeed, Ado inhibits neutrophil release of MMP-9, a proteinase known to degrade the myocardial extracellular matrix. The observed effect of Ado on MMP-9 may therefore open new avenues in anti-remodeling therapy and the effect of Ado and Ado analogs on left ventricular remodeling merits further investigation.
Acknowledgments
We thank Céline Jeanty, Malou Gloesener, and Loredana Jacobs for expert technical assistance, and Nicolaas Brons for his technical help with the flow cytometer.
Sources of Funding
This work was supported by the Centre de Recherche Public-Santé, Luxembourg, and by the Association pour la Recherche sur les Maladies Cardiovasculaires led by Dr Charles Delagardelle, Luxembourg. E.V. is supported by the Ministère de la Culture, de l’Enseignement Supérieur et de la Recherche, Luxembourg.
Disclosures
None.
Footnotes
Original received February 20, 2006; resubmission received July 6, 2006; revised resubmission received August 1, 2006; accepted August 8, 2006.
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the Laboratory of Cardiovascular Research (I.E., D.R., E.V., Y.D.), CRP-Santé, Luxembourg
the Division of Cardiology (D.R.W.), Centre Hospitalier Luxembourg.
Abstract
Matrix metalloproteinases (MMPs), and in particular MMP-9 secreted by neutrophils, are capable of degrading the matrix components of the heart and are thought to be the driving force behind myocardial matrix remodeling after infarction. Adenosine, a naturally produced nucleoside, has been shown to have cardioprotective effects and to inhibit secretion of various cytokines. The aim of our study was to determine the effect of adenosine on the secretion of MMP-9 by neutrophils. Neutrophils were isolated from healthy volunteers through Ficoll and Dextran sedimentation. Neutrophils were activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence or absence of adenosine or adenosine analogs. Zymography and enzyme linked immunosorbent assay were used to measure MMP-9 secretion. Adenosine (1 μmol/L) decreased the fMLP-induced MMP-9 secretion by 30±2% (n=8, P<0.001). The effect was dose-dependent and was not specific to fMLP because adenosine also inhibited MMP-9 secretion by LPS- or H2O2-stimulated neutrophils. The effect of adenosine was mimicked by the adenosine A2a receptor agonist CGS21680 and was inhibited by both the A2a antagonist SCH5826 and A2a RNA silencing. The A3 agonist IB-MECA moderately decreased fMLP-induced MMP-9 secretion. Agonists and antagonists of the other types of adenosine receptors had no significant effect. Adenosine increased intracellular cAMP concentration and accelerated the return to baseline of the intracytoplasmic calcium peak. The inhibition of MMP-9 secretion by adenosine, as well as the calcium effect, was prevented by the protein kinase A inhibitor H-89. In conclusion, we show here that adenosine inhibits MMP-9 secretion by neutrophils. Our results suggest that this effect implies the A2a receptor and is mediated through the cAMP/PKA/Ca2+ pathway. Therefore, adenosine may represent a new approach to prevent matrix degradation and remodeling after myocardial injury.
Key Words: adenosine neutrophils myocardial remodeling protein kinase A matrix metalloproteinase-9
Introduction
Despite reperfusion therapy with thrombolysis or percutaneous intervention, the development of left ventricular remodeling and heart failure after myocardial infarction has not significantly decreased and remains associated with a more than 10-fold elevated risk of death.1 Enhanced activity of matrix metalloproteinases (MMPs) and in particular MMP-9 after infarction breaks down collagen contributing to myocyte slippage and ventricular remodeling. Recent observations2 and the Framingham study have shown that levels of MMP-9 are elevated after myocardial infarction both in the acute and chronic phases.3 We have identified MMP-9 as a risk marker for the development of left ventricular remodeling and heart failure after acute myocardial infarction.4
Animal studies have shown that ventricular remodeling after infarction can be attenuated through the use of MMPs inhibitors.5–7 However, recent results from the PREMIER (Prevention of Myocardial Infarction Early Remodeling) trial failed to demonstrate a beneficial effect of MMP inhibition on left ventricular remodeling after myocardial infarction.8 Therefore, understanding the cellular and molecular triggers of MMPs expression is of major interest to understand and possibly modify the remodeling process.
The nucleoside adenosine (Ado) is formed in the myocardium through dephosphorylation of AMP when there is an imbalance between tissue oxygen supply and demand which occurs in the setting of acute myocardial infarction and congestive heart failure. Multiple lines of evidence have shown that Ado protects against ischemia/reperfusion injury.9 In animals and in humans, Ado can reduce infarct size and improve coronary flow.10 We have shown that Ado inhibits the expression of TNF- in the rat and failing human heart.11,12 However, the effect of Ado on enzymes participating in extracellular matrix degradation has not been studied.
As high levels of MMP-9 are associated with a deleterious outcome after myocardial infarction, the aim of the present study was to investigate the effect of Ado on the expression and secretion of MMP-9. Using near-infrared fluorescent imaging, Chen et al have elegantly demonstrated that infiltrating neutrophils are the likely source of MMP-9 activity early after infarction.13 Therefore, we used isolated neutrophils to study the effect of Ado and Ado analogs on MMP-9. Taken together, our results suggest that pharmacological inhibition of MMP-9 through Ado may favor the stabilization of the extracellular matrix after myocardial infarction.
Materials and Methods
An expanded Materials and Methods section can be found in the supplement available at http://circres.ahajournals.org.
Cell Culture
Neutrophils from healthy donors were isolated from heparinized blood by Ficoll-Hypaque (ICN) centrifugation followed by dextran sedimentation (Amersham, Rosendaal Netherland). Purity of the cell preparations was >93% as determined with a BD FACSCanto flow cytometer (Beckton Dickinson, Erembodegem, Belgium) using anti-CD16 for neutrophils (Immunotools, Friesoythe, Germany). Cell viability, as determined by trypan blue exclusion, was >98%. Purified neutrophils were resuspended at a density of 0.5x106 cells/mL in RPMI 1640 medium containing L-glutamine (Cambrex, Verviers, Belgium) and supplemented with nonessential amino acids and sodium pyruvate. One ml of the cell suspension was seeded per well into 24-well plates. Cells were allowed to rest for 2 hours at 37°C-5% CO2. If indicated, neutrophils were primed with TNF- (10 U/mL) for 15 minutes before preincubation. Cells were preincubated for 5 minutes with Ado (0.01 to 25 μmol/L), Ado receptors agonists (0.1 to 100 nmol/L), or Ado receptors antagonists (1 to 100 nmol/L) as needed. Then fMLP (10–7mol/L), LPS (10 ng/mL), or H2O2 (10 mmol/L) were added and cells were incubated for another 15 minutes (H2O2 and fMLP), or 2 hours for conditions with LPS. For experiments involving siRNAs, neutrophils were transfected using the Nucleofection technology (Amaxa, Cologne, Germany): 1.4 μg of each siRNA was transfected into 1x106 cells following the manufacturer’s recommendations. Controls underwent the same transfection procedure. Cells were incubated for 48 hours before treatments with Ado and fMLP.
Monocytes were purified from peripheral blood mononuclear cells (PBMCs) obtained by Ficoll gradient using the Monocyte Isolation Kit II from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). Purity was estimated by flow cytometry with anti–CD14-PE (monocytes/macrophages), anti–CD13-APC (monocytes and granulocytes), anti–CD20-PE Cy7 (B cells), anti–CD27-FITC (T cells), CD34-PerCP (bone marrow-derived progenitor cells), and anti–CD45-APC Cy7 (leukocytes). Purity was typically above 93%. Monocytes were cultured in RPMI 1640 containing L-Glutamine, sodium pyruvate, nonessential amino acids, penicillin/streptomycin, and 1% fetal calf serum. Differentiation was achieved with 50 ng/mL macrophage-colony stimulating factor (M-CSF) for 7 days. Cells were then preincubated with 10 μmol/L Ado and 10 μmol/L erythro-9-(2-Hydroxy-3-nonyl)-adenine hydrochloride (EHNA, an adenosine deaminase inhibitor) for 15 minutes before activation with 100 ng/mL LPS for 24 hours.
For all cells, conditioned medium was collected, mixed with bovine serum albumin (0.2% final concentration) and protease inhibitors (Roche, Mannheim, Germany), and centrifuged at 600g for 5 minutes, 4°C. Cell-free supernatants were stored at –20°C until use.
Analysis of Gelatinase Activity
Gelatin zymography was performed on cell-free conditioned medium as described before.14 Densitometry was performed on scanned zymograms using the Scion Image program (Scion Corp, Frederick, Mass). The area of the pro–MMP-9 band was used to evaluate gelatinolytic activity, expressed as a percentage of the control condition as indicated. We had previously verified that this band was representative of the global MMP-9 activity (proMMP-9, lipocalin-MMP-9 complex, MMP-9 dimers). A strong correlation was observed between the intensities of the three bands.
Measurement of cAMP Concentration
cAMP concentration was assessed using the direct cAMP enzyme immunoassay kit according to the manufacturer’s protocol. Because of low cAMP concentrations, 3x106 cells were incubated in 6-well plates and the acetylated version of the kit was used. Detection limit of the assay is 0.037 pmol/mL.
Measurement of the Intracellular Calcium Concentration
For these experiments, Fluo-4/AM was used as the fluorescent, Ca2+-sensitive indicator. Neutrophils (1x106 ml–1) were preloaded with Fluo-4/AM (1 μmol/L) and Pluronic (0.02%) at room temperature on a shaker in Hanks balanced salt solution (HBSS, pH 7.4). After a 30-minute incubation, cells were washed twice in indicator-free HBSS and then incubated in HBSS for a further 15 minutes at room temperature in presence or absence of H89 (10 μmol/L). 0.5x106 cells were transferred to 96 wells plate or BD Falcon tubes (5 mL) for analysis at 37°C using a POLARstar OPTIMA fluorescence spectrophotometer (ex 490 nm and em 520 nm) and a BD FACSCanto flow cytometer (ex 488 nm and em 530/30 nm), respectively. After a stable baseline was obtained (1 minute), the neutrophils were incubated with or without Ado (1 μmol/L) during 1.5 minutes and finally were activated by addition of fMLP (10–7M). The subsequent increase in Fluo-4/AM fluorescence intensity was monitored more than a 5 minutes period.
Cytotoxicity Assay
To determine a potential cytotoxicity of any of the drugs used in our experiments, cell supernatants were assayed for lactate dehydrogenase (LDH) activity using a LDH-based cytotoxicity detection kit (Roche) as described by the manufacturer. None of the drugs used induced a significant cytotoxicity.
Data Analysis
The results are shown as mean±SEM. Most of the experiments are the mean of 3 independent experiments, unless otherwise stated. Statistical analysis was performed after passing a normality test through the GraphPad Instat program, using one-way t test or one-way ANOVA, and P<0.05 was considered to be significant.
Results
Adenosine Inhibits MMP-9 Secretion By Neutrophils
Neutrophils were preincubated for 5 minutes with Ado (0.01 to 25 μmol/L) or medium alone before fMLP stimulation for 15 minutes. Gelatinase activity was measured in cell-free conditioned medium by zymography. As shown in Figure 1A, three forms of MMP-9 were detected: pro–MMP-9, MMP-9-lipocalin complex, and pro–MMP-9 homodimer, an observation consistent with previous publications.15 Addition of fMLP induced a 2-fold increase of MMP-9 secretion as compared with control conditions. Whereas Ado had no effect on basal MMP-9 secretion (not shown), it significantly decreased all 3 forms of MMP-9 in a concentration-dependent manner in fMLP-treated neutrophils. The maximal effect of Ado was observed at 1 μmol/L with a 30±2% (n=8, P<0.001) inhibition of pro–MMP-9 secretion (Figure 1A). All 3 forms of MMP-9 were decreased to the same extent. The results obtained by zymography were confirmed by ELISA (Figure 1B). Of note, an Ado concentration of 1 μmol/L can be observed in vivo in conditions like heart failure16 and sepsis.17
Because neutrophils are likely to be primed in the context of myocardial injury, we verified that Ado was able to inhibit MMP-9 secretion by neutrophils primed with TNF- before treatment. Priming did not affect the response of neutrophils to Ado and fMLP (results not shown).
Although 15 minutes of stimulation with fMLP is likely too short to induce a de novo synthesis of the MMP-9 protein, PCR was performed and revealed that neither fMLP nor Ado had an effect on MMP-9 mRNA expression in our experimental conditions (results not shown). This observation confirmed that the increase of MMP-9 in cell-culture supernatants is solely attributable to neutrophil degranulation. To test whether the Ado effect was limited to MMP-9 (tertiary granules), culture supernatants were assessed for MMP-8 (secondary granules) and MPO (primary granules) contents. Figure 1C clearly shows that Ado also reduces the fMLP-induced release of MMP-8 and MPO, arguing for a non granule-specific effect of Ado.
The Inhibition Is Not fMLP-Dependent, and Is Adenosine-Related
To verify whether the inhibition of MMP-9 secretion by Ado was specific to fMLP, LPS and H2O2 were used as additional stimulators of MMP-9 secretion. Neutrophils were preincubated for 5 minutes with Ado (1 μmol/L) or medium before stimulation with either LPS (10 ng/mL) or H2O2 (10 mmol/L). 1% autologous serum was added to the culture medium used for LPS experiments. Similarly to fMLP-treated cells, Ado inhibited MMP-9 secretion by neutrophils stimulated with LPS or H2O2 (Figure 2A). This observation indicated that the effect of Ado on MMP-9 secretion was not restricted to the bacterial agents fMLP or LPS but also occurs when cells are stimulated with a reactive oxygen species generator such as H2O2.
The so-called Ado modulators ITU (an inhibitor of Ado kinase) and DIP (an inhibitor of Ado transport) are substances known to increase the endogenous concentration of Ado. We tested their ability to replicate the inhibition of MMP-9 secretion by Ado. Both adenosine modulators reproduced the effect of Ado (Figure 2B). Furthermore, replacing Ado by the nonspecific Ado receptor agonist NECA resulted in a similar inhibition of MMP-9 secretion (Figure 2B). These data reinforced our finding that Ado is a reproducible inhibitor of MMP-9 secretion.
Inhibition of MMP-9 Secretion by Ado Is Mediated by the A2a Receptor
To evaluate the type of Ado receptor involved in the inhibition of MMP-9 secretion by Ado, a pharmacological approach using several Ado receptors agonists and antagonists was initially chosen. The A2a-specific agonist CGS21680 was able to replicate the inhibitory effect of Ado on MMP-9. The A3-specific agonist IB-MECA showed a weaker effect on MMP-9 at high concentration whereas the A1 specific agonist C8031 was essentially without effect (Figure 3A). Among the 4 Ado receptors antagonists tested, only the A2a-specific antagonist SCH58261 was able to suppress the effect of Ado on MMP-9 (Figure 3B). To rule out the possibility that this suppression could be a result of an increase in MMP-9 secretion by SCH58261 itself, additional experiments were performed. The A2a antagonist SCH58261 at the concentration of 100 nM used in these experiments and giving maximal inhibition of MMP-9 release did not modify the fMLP-induced MMP-9 secretion (results not shown).
To compensate for a potential lack of specificity of the Ado agonists and antagonists, a second approach taking profit of the highly specific RNA interference process was used. Neutrophils were transfected with siRNA specific for the A2aAR or A3AR receptors before treatment with Ado and fMLP. Figure 3C shows that only the A2aAR-specific siRNA reversed, at least partly, the inhibitory effect of Ado on MMP-9 secretion. Taken together, these results demonstrate that the inhibition of MMP-9 secretion by Ado is most probably mediated by the A2aAR.
Human Neutrophils Express Both A2aAR and A3AR mRNA
The relative expression of the 4 different Ado receptors on neutrophils is poorly characterized. Using quantitative PCR, we confirmed the presence of both the A3 and the A2a receptors on neutrophils. The absence of any amplified products in a control reaction without reverse transcriptase (-RT) attested the absence of any contaminating genomic DNA in the RNA preparations (Figure 4A). Quantification revealed that the A2a receptor was 2.5-fold higher expressed than the A3 receptor (Figure 4B). These data are in accordance with the results obtained with Ado receptor agonists and antagonists, ie, the A2a receptor appears to be the key mediator of the inhibition of MMP-9 secretion by Ado.
The Inhibition of MMP-9 Secretion by Ado Is cAMP-, PKA-, and Calcium-Related
The bacterial agent fMLP is known to trigger neutrophil degranulation through a massive inositol triphosphate-mediated calcium release from intracellular Ca2+ stores. We investigated whether the cAMP/PKA pathway could be responsible for the observed effect of Ado through modulation of intracellular Ca2+ stores. The effect of Ado on intracellular cAMP in unstimulated and fMLP-stimulated neutrophils is shown in Figure 5. Ado increased cAMP in unstimulated and fMLP-stimulated cells to the same extent.
Because cAMP formation leads to PKA phosphorylation, the involvement of PKA in the effect of Ado was evaluated using the specific PKA inhibitor H89. When added to untreated neutrophils or fMLP-treated neutrophils, H89 did not modify MMP-9 secretion. However, it abrogated the inhibition of MMP-9 secretion by Ado (Figure 6).
Finally, the kinetic of free cytosolic calcium concentration was studied less than fMLP stimulation, in the presence or absence of Ado. Figure 7A shows a representative pattern of Fluo-4 responses in fMLP-activated neutrophils obtained by spectrofluorimetry. The addition of fMLP to neutrophils triggered the characteristic, abrupt increase in Fluo-4 fluorescence because of elevated concentrations of cytosolic Ca2+. Ado did not modify the magnitude of this increase but accelerated the rate of subsequent decline in fluorescence intensity, indicating an accelerated clearance of Ca2+ from the cytosol. Figure 7B shows that Ado shortens the time to half-peak from 43.7±11.4 sec to 23.5±6.3 sec (P<0.001), an effect totally reversed by adding H89. These results were confirmed by flow cytometry (not shown). Moreover, a reproductive decrease of cytosolic calcium by 24±7% (P<0.001) was observed during the preincubation of the neutrophils with Ado before stimulation with fMLP, indicating that Ado has an effect per se on cytosolic calcium. Signal transduction pathways activated by fMLP in human neutrophils have been recently reviewed and include the protein kinase C (PKC) pathway.18 Using the PKC inhibitors chelerythrine and BIS10, we ruled out any involvement of the PKC pathway in the inhibition of MMP-9 secretion by Ado (results not shown).
Ado Does Not Inhibit MMP-9 Secretion by Monocytes/Macrophages
Although neutrophils constitute the first wave of inflammatory cells recruited to the infarcted area, other cell types are operative in the context of myocardial injury, such as macrophages and fibroblasts. We tested whether Ado modifies the extent of MMP-9 secretion by cells from the monocytes/macrophages lineage. The effect of Ado was studied in monocytes, LPS-activated monocytes, and M-CSF–differentiated monocytes. On stimulation, MMP-9 release was gradually increased with the degree of activation and maturation of monocytes. However, in contrast to neutrophils, Ado did not significantly modify MMP-9 release in monocytes, LPS activated monocytes, or M-CSF–differentiated monocytes (Figure 8).
Discussion
In this study, we demonstrate for the first time that Ado inhibits fMLP-induced MMP-9 secretion in human neutrophils. We also provide evidence that the mechanism of the inhibitory effect of Ado on the fMLP-induced MMP-9 secretion is mediated by the A2aAR through a cAMP-PKA pathway and acceleration of Ca2+ clearance from the cytosol of activated neutrophils.
Ado has been shown to attenuate major neutrophil functions including cell adhesion, superoxyde anion production, and phagocytosis, effects mainly dependent on activation of the A2aAR. However, the effect of Ado on neutrophil degranulation has been only partially described. Ado inhibits the secretion of lysozyme,19 myeloperoxidase,20 and elastase,21 3 enzymes stored in the primary granules. The effect of Ado on gelatinase granules or tertiary granules involved in matrix degradation and diapedesis of neutrophils22 has not been studied. Gelatinase granules contain MMP-9, a member of the MMPs family known to play a pivotal role in the degradation of type IV collagen in basement membranes during left ventricular remodeling.
Our results confirm the presence of the A2aAR on neutrophils and show that A3 receptors are also present on neutrophils. So far, the A3AR protein has only been identified in human eosinophils23 and monocytes24 and in a rat basophil leukemia cell line,25 despite the detection of A3AR gene transcripts in a variety of species and tissues. Pharmacological studies using agonists and antagonists identified A3AR in human neutrophils and promyelocytic HL-60 cells.26–29
Using both pharmacological (Ado agonists and antagonists) and molecular (siRNAs) tools, we were able to show that Ado inhibits the secretion of MMP-9 mainly through its A2aAR. A small participation of the A3AR cannot completely be excluded. It has to be kept in mind that the A3 agonist used, IB-MECA, may not be as specific as previously thought. Indeed, data from a recent report suggest that IB-MECA may also bind to the A2aAR, which may explain some of the effects we observed with that compound.30 A limitation of the experiments involving RNA interference is related to the weak transcriptional machinery of neutrophils. However, our data show that cells transfected with the A2aAR-specific siRNA reproducibly lacked the inhibitory action of Ado on MMP-9 secretion.
In general, occupancy of A2aAR stimulates accumulation of intracellular cAMP, which acts as a second messenger to alter cellular functions.27 Our results suggest that the intracellular mechanism responsible for the inhibition of MMP-9 secretion by Ado involves cAMP, PKA, and intracellular calcium stores. This is supported by the fact that cAMP concentrations increase significantly after the addition of Ado and by the observation that the specific PKA inhibitor H89 completely suppresses the effect of Ado on MMP-9. The rise in cAMP after addition of Ado has been well documented.31,32 Because adenylate cyclase is activated by Gs protein, the increase in cAMP is consistent with involvement of A2aAR, a Gs-coupled receptor.
Intracellular calcium increases after neutrophil stimulation,33 leading to degranulation. On the other hand, it is known that PKA is associated with a decrease in intracellular calcium, possibly through activation of phospholamban and/or sarcoplasmic reticulum–associated Ca-ATPase. We show here that Ado does not alter the peak calcium levels but markedly accelerated the normalization of calcium levels after stimulation. This is associated with significant inhibition of MMP-9. Our study is in accordance with recent reports showing that the A2aAR may antagonize proinflammatory stimuli and restore calcium homeostasis in neutrophils.26,34
The action of Ado highlighted in this article appears to be restricted to certain cell types participating in the process of myocardial injury and remodeling. Whereas Ado reproducibly inhibited MMP-9 secretion by neutrophils, it had no effect on other inflammatory cells such as monocytes/macrophages. This is in line with recent data from Spinale et al35 showing a reduction in left ventricular remodeling where MMP activity is inhibited early on after myocardial infarction but not at a later stage. Thus, there appears to be a time window for MMP inhibition after infarction.
In conclusion, our results suggest that, in addition to the known effects of Ado on myocardial reperfusion injury and infarct size, Ado may also have a beneficial effect at the level of the extracellular matrix. Indeed, Ado inhibits neutrophil release of MMP-9, a proteinase known to degrade the myocardial extracellular matrix. The observed effect of Ado on MMP-9 may therefore open new avenues in anti-remodeling therapy and the effect of Ado and Ado analogs on left ventricular remodeling merits further investigation.
Acknowledgments
We thank Céline Jeanty, Malou Gloesener, and Loredana Jacobs for expert technical assistance, and Nicolaas Brons for his technical help with the flow cytometer.
Sources of Funding
This work was supported by the Centre de Recherche Public-Santé, Luxembourg, and by the Association pour la Recherche sur les Maladies Cardiovasculaires led by Dr Charles Delagardelle, Luxembourg. E.V. is supported by the Ministère de la Culture, de l’Enseignement Supérieur et de la Recherche, Luxembourg.
Disclosures
None.
Footnotes
Original received February 20, 2006; resubmission received July 6, 2006; revised resubmission received August 1, 2006; accepted August 8, 2006.
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