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Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: (1) attraction of phagocytes via soluble `find me' signals, (2) recognition and phagocytosis via cell surface presenting `eat me' signals, and (3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of pro-inflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the present study, using wild type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, possibly via activation of activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase / protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.
Most cell types have a limited life span, which ends physiologically through the process of apoptosis, or programmed cell death. In vivo, apoptotic cells are usually engulfed by neighboring cells or professional phagocytes, such as macrophages (1). As they become apoptotic, cells undergo dramatic changes in the composition of their surface, which allows their recognition by phagocytes and subsequent removal. Apoptotic cell clearance is believed to represent a critical process in tissue remodeling, maintenance of immune homeostasis, and resolution of inflammation (2, 3).
While the phagocytosis of a variety of pathogenic targets, especially bacteria and virally-infected cells, normally triggers a pro-inflammatory response in macrophages (including the generation of reactive oxygen-derived intermediates, the release of proteolytic enzymes, and the production of numerous inflammatory cytokines), ingestion of apoptotic cells by macrophages usually induces an anti-inflammatory phenotype. Apoptotic cells do not simply fail to provide pro-inflammatory signals; rather, they actively interfere with the inflammatory program. For example, preincubation of macrophages with apoptotic cells strongly suppresses the inflammatory response induced via Toll-like receptor 4 by lipopolysaccharide, a component of the cell wall of Gram-negative bacteria (4–6). This inhibitory property appears to be a common attribute acquired post-translationally by all cells undergoing apoptotic cell death, regardless of the cell type or the particular death stimulus (6–8).
The mechanism(s) by which apoptotic cells exert their inhibition on phagocytes may vary over time. The earliest anti-inflammatory activity of the apoptotic corpse is manifest as an immediate-early inhibition of macrophage pro-inflammatory cytokine gene transcription and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement (6). This transcriptional inhibition is evident, for example, on the level of NF-κB-dependent transcription, and occurs without effect on proximal signaling events induced by inflammatory receptors, including innate immune receptors of the Toll like receptor family (6). In case of IL-12 for example, cell-cell contact with apoptotic cells is sufficient to induce the inhibition of the cytokine production by activated macrophages via a novel zinc finger nuclear factor, GC-BP, which selectively inhibits IL-12 p35 gene transcription (9).
Subsequently, soluble mediators may act in a paracrine or autocrine fashion to amplify and sustain the anti-inflammatory response. For example, Voll et al. observed an inverse relationship between the secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-12, and the transient release of IL-10 from lipopolysaccharide-stimulated macrophages following their interaction with apoptotic cells (4). Fadok et al. reported a similarly inverse relationship between the release of transforming growth factor (TGF)-β, platelet-activating factor, and prostaglandin E2 and the secretion of TNF-α, IL-1β, and several other inflammatory cytokines by macrophages following engulfment of apoptotic targets (5). Although the pharmacologic blockade of platelet-activating factor and prostaglandin E2 signaling had only slight effect (10), the addition of TGF-β-specific neutralizing antibody to cultures of macrophages and apoptotic targets partially restored pro-inflammatory cytokine release (5, 10) indicating a central role of this cytokine in mediating anti-inflammatory responses.
Adenosine is a purine nucleoside that, following its release from cells or after being formed extracellularly, diffuses to the cell membrane of surrounding cells, where it binds to its receptors (11, 12). There are four adenosine receptors, all of which are G protein-coupled receptors and are abundantly expressed by macrophages (13). The genes for these receptors have been analyzed in detail and are designated A1, A2A, A2B and A3. Adenosine A1 receptors are stimulated by 10−10−10−8 M concentrations of adenosine and mediate decreases in intracellular cyclic AMP (cAMP) levels, adenosine A2A and A2B receptors are stimulated by higher (5 × 10−7 M and 1 × 10−5 M, respectively) concentrations of adenosine and mediate increases in cAMP levels, while adenosine A3 receptors are stimulated by 10−6 M concentrations of adenosine and mediate adenylate cyclase inhibition (12). Although adenosine is constitutively present in the extracellular space at low concentrations (<1 μM), its concentration can dramatically increase under inflammatory conditions reaching concentrations high enough (14, 15) to exert immunomodulatory and especially immunosuppressive effects (13).
Since many of the immunosuppressive effects of adenosine on macrophages have been reported to be mediated via adenosine A2A receptors (A2ARs)3 (13), we asked whether adenosine acts as a soluble mediator, via A2ARs, to prevent pro-inflammatory cytokine production by macrophages engulfing apoptotic cells.
All reagents were obtained from (Sigma-Aldrich, Budapest) except indicated otherwise.
Most of the experiments were done using 3 months old male wild type or adenosine A2A receptor deficient mice (16) generated on an FVB background. Some of the experiments were also carried out on Peroxisome Proliferator-Activated Receptor (PPAR)δ (17) or liver X receptor (LXR) (18) deficient mice generated on 129 SvJ, or on a mixed background of C57Bl/6 and 129Sv, respectively. These studies have been reviewed and approved by the review committee of the University of Debrecen [DEMÁB].
Macrophages were obtained by peritoneal lavage with sterile physiological saline. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 1 mM Napyruvate, 50 μM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2 for 2 days before use. After 3–4 hrs. incubation, the non-adherent cells were washed away. Before the experiments the cells were cultured for 2 days replacing media daily. For bone marrow-derived macrophages, wild type and PPARδ null bone marrow was isolated from femurs, and cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 1 mM Na-pyruvate, 50 μM 2-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin and 10% L929 conditioned media for 10 days. The non adherent cells were washed away from the 3rd day daily.
Apoptotic cells were prepared from wild-type mice in all cases. Thymocytes isolated from 4 weeks old mice were cultured for 24 hrs. (107 cells/ml) in RPMI 1640 medium supplemented with penicillin/streptomycin in the absence of serum. In case of NB4 cells the apoptosis was induced by 10 μM As2O3 –treatment for 12hrs (19). This method typically resulted in >80% apoptotic cells (as assessed by propidium iodide/AnnexinV-FITC staining). Apoptotic cells were used at a 10:1 (apoptotic cell: macrophage) ratio.
The experiments were performed by co-incubating A2AR+/+ macrophages (1 × 106 cells / sample) with apoptotic cells in 1:10 ratio. For the respective experiments, macrophages were pre-treated with 50 mM cytochalasin D for 1 hr. to block the phagocytic activity of macrophages.
After 2 hrs. of phagocytosis the supernatants were replaced with fresh culture media. After 5 hrs. of incubation (at 37°C) the supernatants were collected, deproteinized with 5 ml ice cold 0.6 N HClO4 and stored on −80°C. The determination of adenosine concentration was carried out with a reverse phase HPLC method as described (20).
Wild type and A2AR null peritoneal macrophages were coincubated with apoptotic thymocytes for one hr. in 1:10 ratio. After replacing media and washing away the apoptotic cells, macrophages were incubated for additional one, three or five hrs. For some experiments all these treatments were carried out in the presence of 5 μg / ml actinomycin D, or 0.1 μg / ml cycloheximide. For characterizing the regulation of the expression of the receptor macrophages were treated with various concentrations of 22-R(OH)-cholesterol, an LXR agonist, or GW501516, a PPAR δ agonist (Glaxo Smith Kline), for 3 hrs. After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control. For the detection cells were stained with FITC-conjugated anti-goat IgG. Stained cells were analyzed on a FACSCalibur (BD Biosciences). The results were analyzed by WinMDI 2.9 software.
Wild type, A2AR null and LXR null peritoneal, or PPARδ wild type and knock out bone marrow derived macrophages were coincubated with apoptotic thymocytes for one hr. in 1:10 ratio for 1 hr. After washing away the apoptotic cells and replacing media mRNA was collected 2 hours later.
Wild-type and A2AR null peritoneal macrophages were plated onto 24-well plates at a density of 5 × 105 cells / well. To determine cytokine production by macrophages exposed to apoptotic cells, macrophages were exposed to apoptotic cells for one hr in the presence or absence of an A2AR-selective antagonist SCH442416 (10 nM, Tocris). Apoptotic cells than were washed away, the SCH442416 was re-added and the macrophages were cultured for an additional five hrs. in fresh RPMI-1640 media (10% FBS). At the end of culture cell culture media were analyzed by Mouse Cytokine Array (Proteome Profile Array from R&D Systems). The pixel density in each spot of the array was determined by Image J software. Alternatively, cytokine-induced neutrophil-attracting chemokine (KC), TGF-β, macrophage inflammatory protein-2 (MIP-2) and IL-10 cytokine levels were measured with R&D Systems ELISA kits.
Total RNA was isolated from control and treated macrophages (3 × 106 cells / sample) by TRI reagent. After various treatments 3 × 106 peritoneal macrophages were washed with ice-cold PBS. RNA was extracted with Tri-reagent. cDNA was synthesized with High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instruction. Cyclophilin, MIP-2, adenosine A2A receptor, nitrogen monoxide synthetase (iNOS and eNOS), arginase I and II levels were determined with Taq-Man PCR using FAM-GMB-labelled primers (Applied Biosystems). Real-time PCR plates were run on an ABI Prism 7900 using ABM Prism SDS2.1 software for evaluation (Applied Biosystems). Samples were run in triplicate. Gene expression was normalized to cyclophilin expression.
For phagocytosis assays, macrophages were stained overnight with10 μM 5-(and 6-)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR) (Invitrogen), while thymocytes were labeled overnight with 6 μM 6-carboxy-3′, 6′-diacetylfluorescein (CFDA) (Invitrogen). Macrophages were incubated with apoptotic thymocytes in 10:1 target/macrophage ratio for one hr. Cells incubated with apoptotic thymocytes incubated at 4 °C were used as controls. After washing, the cells on the plate were trypsinized, resuspended in cold medium with 0.5% sodium azide, and 10,000–20,000 cells were analyzed for each point by two-color flow cytometry (FACSCalibur, BD Biosciences).
For visualizing apoptotic cells in macrophages, macrophages were plated in 2-well chamber slides in a concentration of 5×105/well and cultured for 48 h before CMTMR staining. After coculturing macrophages with CFDA labeled apoptotic cells for 30 min, cells were washed and fixed in ethanol/acetone (1:1) for 10 min at −20°C. Images were taken with a Olympus FV1000 confocal laser scanning microscope.
A2AR+/+ or A2AR−/− macrophages were exposed to apoptotic cells for 1 hr. Media were replaced and macrophages were incubated for an additional 1 hr. Cell culture supernatants were analyzed for NO production by measuring nitrite, a stable oxidation product of NO, using the Griess-Ilosvay method (21).
A2AR+/+ and A2AR−/− mice were injected with 2 ml of 4% thioglycollate intraperitoneally. 4 days later they were injected intraperitoneally with 2× 106 of apoptotic cells suspended in 2 ml of physiological saline or 2 ml physiological saline. After 3 hrs. the peritoneal cells were collected, washed, blocked with 50% FBS and stained with FITC-conjugated rat anti-mouse Gr-1 (RB6–8C5) (Pharmingen), or V450 rat IG2bκ isotype control for 30 min. The detection was carried out using FITC conjugated anti-rat IgG (Pharmingen). Cells were then washed, fixed and analyzed by flow cytometry (FACSCalibur, BD Biosciences) to determine the percentages of neutrophils in the total cell population. In some experiments rat anti–mouse KC (clone 48415.111; IgG2a), rat anti–mouse MIP-2 (clone 40605; IgG2b) or their isotype controls IgG2a, (clone 5444.11), and IgG2b (clone 141945) obtained from R&D Systems were injected together with the apoptotic cells into A2A−/− mice.
All the data are representative of at least three independent experiments carried out on three different days. Values are expressed as mean ± S.D. P values were calculated by using two-tailed Student's t-test for two samples of unequal variance. The analysis of cytokine array experiments was carried out by ANOVA test. Statistical significance is indicated by a single asterisk (P < 0.05).
First we tested whether macrophages engulfing apoptotic cells for 2 hrs. release adenosine during the following 5 hrs. While the adenosine levels in the culture medium alone, or the culture medium of macrophages or thymocytes cultured alone for the same time period were below the detection limit (0.1–0.25 μM), adenosine was found in the culture medium of macrophages exposed previously to apoptotic cells (3.34 ± 1.4 μM, n=3 independent experiments). Induction of the release of adenosine was not specific for thymocytes as apoptotic targets, such as uptake of As2O3-treated apoptotic NB4 acut promylocytic leukemia cells (19) also resulted in adenosine release (5.1 ± 1.4 μM, n=3). This concentration of adenosine is in the range of the adenosine A2A receptor sensitivity. Inhibition of phagocytosis by cytochalasin D, an inhibitor of actin polymerization, completely prevented the appearance of adenosine, indicating that the production is dependent on apoptotic cell engulfment.
As shown in Figure 1A, mouse peritoneal macrophages engulfing apoptotic cells not only produce adenosine, but also express A2ARs, and this expression is significantly enhanced following incubation with apoptotic cells. Preincubation of macrophages for 30 min. with actinomycin D, a transcription inhibitor, or cycloheximide, a protein synthesis inhibitor, prevented the apoptotic cell-associated induction of adenosine receptor expression, indicating that regulation occurs on the transcriptional level (Fig. 1B). Indeed, induction on the level of mRNA was evident following the engulfment of apoptotic cells (Fig.1C).
Cytochalasin D does inhibit the engulfment process, but it does not influence the recognition of apoptotic cells (6). Binding of phosphatidylserine on the surface of apoptotic cells plays a role in their recognition and subsequent uptake by macrophages, and this recognition can be inhibited by preincubation of apoptotic cells with recombinant annexin V (which binds to phosphatidylserine; ref. 22). Both cytochalasin D and recombinant annexin V inhibited the induction of A2AR expression by apoptotic cells (Fig. 1C and D) suggesting that it is the engulfment of apoptotic cells, rather than their recognition per se, that triggers enhanced A2AR expression.
Recently, two lipid-sensing nuclear receptors (LXR and PPARδ) expressed by macrophages have been implicated in promoting phagocytosis and anti-inflammatory effects of apoptotic cells following their uptake (17, 18). Both 22-(R)OH-cholesterol, an LXR agonist, and GW501516, a PPARδ agonist, promoted the mRNA expression of A2AR in peritoneal macrophages (Fig.1 E and F) indicating that both LXR and PPARδ might mediate the effect of apoptotic cell engulfment on A2AR expression. Since the effect of these agonists might be not be fully specific, to prove further the involvement of these receptors in the A2AR upregulation LXR knock out and PPAR delta knock out macrophages were also tested for their response. While in case of PPAR delta knock out macrophages the upregulation of A2AR was significantly attenuated as compared to their wild-type controls (Fig. 1G), we could not draw a definite conclusion from the LXR KO mice, as their wild type control did not show an upregulation (Fig. 1H). We have no explanation why induction of the expression of adenosine A2A receptor was seen in macrophages on FVB and 129/SvJ backgrounds, but was not seen in mice on a mixed background of C57Bl/6 and 129Sv.
Evaluation of the cytokine secretion profile of unstimulated macrophages was performed using a highly sensitive cytokine antibody array method, enabling the simultaneous detection of low concentrations of multiple cytokines in one assay (picogram per milliliter range). The map of the 40 cytokines detected on the membranes is diagrammed in Figure 2A. The cytokines in our experimental systems were first evaluated by experiments using untreated wild-type and A2AR−/− macrophages in vitro. The results reported in Figure 2B show that ~85% of all available cytokines on the filters were detectable, even though some were at a very low levels. The loss of the A2AR did not affect significantly the level or the composition of most of the cytokines released. However, as shown in Figure 2C, when macrophages were exposed to apoptotic cells, we found 9 cytokines whose production, although not affected in wild type cells, was increased in A2AR−/− macrophages. These cytokines include the interferon-gamma inducible protein 10 kD (IP-10), the cytokine-induced neutrophil-attracting chemokine (KC), and the macrophage inflammatory proteins-1α, −1β and -2 (MIP-1α, −1β, -2), which act as chemoattractants for neutrophils and / or other cell types (23–26). The pro-inflammatory cytokines IL-17 (27) and IL-1α (28), and IL-3, which stimulates the differentiation of multipotent hematopoietic stem cells into the myeloid direction and proliferation of all cells in the myeloid lineage (29), also were produced in an enhanced amount. In addition, release of the antagonist of the IL-1 receptor (IL-1ra) was also enhanced. The induction or the modification in the levels of these cytokines was not due to the presence of high amount of late apoptotic cells or altered phagocytosis, as the majority of apoptotic cells were propidium iodide negative (Fig. 2D), and no differences in the extent of phagocytosis of wild-type and A2AR−/− macrophages were observed (Fig. 2E–G). Among the cytokines released in altered amounts in the supernatant by cultured A2AR−/− macrophages, MIP-2 showed the most dynamic change in response to apoptotic cell exposure, and MIP-2 and KC levels were detected in the highest amounts (Fig. 2B and C). No cytokines were detected in the supernatants when thymocytes (viable or apoptotic) were incubated alone (data not shown), demonstrating that the secreted cytokines were macrophage-related.
To make sure that alterations in the cytokine profile were not a result of adenosine-receptor-related, but developmental effects in the A2A KO vs. wild-type macrophages, cytokine release of wild-type macrophages exposed to apoptotic cells were also tested in the presence of the highly specific A2AR specific antagonist, SCH 442416. As shown in Figure 3A–C, we could confirm the enhanced expression of KC and MIP-2, in these experiments as well indicating that the altered pattern of KC and MIP-2 secretion observed in A2AR−/− macrophages is indeed a consequence of the lack of the adenosine A2A receptor signaling during phagocytosis of apoptotic cells. In addition, the enhanced expression of MIP-1α and MIP-1β, two further neutrophil chemoattractants (26), was more clearly seen (Fig3 A, D and E).
To confirm the results, MIP-2 and KC protein levels also were assessed by ELISA. In harmony with the cytokine array results, although the production of MIP-2 and KC are not altered by wild-type macrophages exposed to apoptotic cells, the release of both MIP-2 and KC by A2AR−/− macrophages is enhanced under the same conditions (Fig 3F and G).
Since we found that, in the absence of adenosine A2A receptors, macrophages release the neutrophil chemoattractant MIP-2 (CXCL-2) and KC (CXCL-1) when they engulf apoptotic cells, we decided to investigate whether in A2AR−/− mice injection of apoptotic cells affects migration of neutrophils in a sterile peritonitis model. For this purpose, mice were injected intraperitoneally, first with thioglycollate and then, 4 days later, with 2× 106 apoptotic cells. As shown in Figure 4A, injection of apoptotic thymocytes did not induce a significant neutrophil migration into the peritoneum in wild-type mice, while in the A2AR−/− mice a significant neutrophil migration was detected in this model. This was accompanied by enhanced levels of MIP-2 and KC in the peritoneal fluid of A2A−/− mice (Fig. 4B). To test whether the enhanced production of KC and MIP-2 together are responsible for the phenomenon, blocking antibodies anti-KC (50 μg) and anti–MIP-2 (50 μg) or their isotype controls were added together with the apoptotic cells. As shown in Figure 4C, addition of blocking antibodies to both KC and MIP-2 completely prevented the migration of neutrophils, while their isotype controls had no effect. Addition of blocking antibodies to MIP-2 alone did not fully block the migration of neutrophils. These data indicate that the loss of adenosine A2A receptors leads to sufficient neutrophil chemoattractant production by macrophages engulfing apoptotic cells to affect migration of neutrophils in an in vivo peritonitis model.
Since among the neutrophil chemoattractants, the release of which was altered by the loss of A2AR, MIP-2 showed the most dramatic changes during phagocytosis of apoptotic cells, we decided to investigate further the alterations in the regulation of this cytokine. Since previous studies indicated that in long term (one day) experiments TGF-β1 and IL-10 might mediate the anti-inflammatory effects of apoptotic cells (4,5,10), we checked whether TGF-β1 or IL-10 release is altered in macrophages lacking A2AR. However, in such short term experiments we could not detect the release of IL-10 neither with the cytokine array nor by the ELISA technique. Active TGF-β was detectable, but its production was not altered in the A2AR−/− macrophages (Fig. 3H). These data indicate that not an altered IL-10 or TGF-β production regulates the altered MIP-2 production in A2AR−/− macrophages. That is why we decided to investigate a possible direct regulation by the A2A receptors.
First we tested the role of the adenylate cyclase pathway, since many of the anti-inflammatory effects of the adenosine A2A receptor were reported to be mediated via this signaling pathway (13). For this purpose we exposed A2AR−/− macrophages to various compounds known to elevate intracellular cAMP levels: cholera toxin, which is known to increase cAMP levels by ADP-ribosylation of the stimulatory G protein of adenylate cyclase (30); forskolin, an adenylate cyclase activator (31); and dibutyryl-cAMP, a membrane permeable variant of cAMP. Preincubation of A2AR−/− macrophages with these compounds for 30 min. prevented the increase in MIP-2 levels when exposed to apoptotic cells (Fig. 5A). On the other hand, preincubation of wild-type macrophages with Rp-cAMPS triethylamine, a specific membrane-permeable inhibitor of activation by cAMP of cAMP dependent protein kinase I and II (32), or recombinant adenosine deaminase, which degrades adenosine, resulted in an increase in MIP-2 production when exposed to apoptotic cells (Fig. 5B). These data together indicate that MIP-2 production by macrophages exposed to apoptotic cells is actively suppressed by the A2AR stimulated by adenosine in an autocrine way using the adenylate cyclase / protein kinase A signaling pathway.
Preincubation of A2AR−/− macrophages for 30 min with actinomycin D, a transcription inhibitor, or cycloheximide, a protein synthesis inhibitor, prevented the apoptotic cell-associated induction of MIP-2 release, indicating that regulation occurs at the transcriptional level (Fig. 5C). Indeed, engulfment of apoptotic cells induced the mRNA levels of MIP-2 in A2AR−/− macrophages, but not in their wild-type counterparts (Fig. 5D).
Previous studies have shown that NO is released by macrophages exposed to early apoptotic cells (33), and that NO can contribute to MIP-2 production (34, 35). To investigate the potential role of NO in the apoptotic cell-induced MIP-2 production in A2AR−/− macrophages, macrophages were exposed to the nitric oxide synthase (NOS) inhibitor L-(G)-Nitro-L-arginine methyl ester (L-NAME) before the addition of apoptotic cells. Addition of L-NAME attenuated MIP-2 protein (Fig. 6A) and mRNA (Fig. 6B) expression in both wild-type and A2AR−/− macrophages induced by exposure to apoptotic cells, indicating that NO production contributes to the effect. We further investigated whether NO production is altered in the absence of adenosine A2A receptor. As shown in Figure 6C, in accordance with previous reports (34), macrophages exposed to apoptotic cells produce NO. Indeed, apoptotic cell-induced NO production was enhanced in A2AR−/− macrophages, as compared to their wild-type counterparts. Since the addition of sodium nitropusside, a potent NO donor, enhanced apoptotic cell-induced MIP-2 production in wild type macrophages (Fig 6D), our data indicate that NO contributes to the apoptotic cell-induced MIP-2 production. However, addition of sodium nitropusside to macrophages alone was not able to induce MIP-2 production, implying that apoptotic cell-derived signals contribute to the induction of MIP-2 (Fig 6D).
Similar to the induction of MIP-2 (Fig. 5A and B), production of NO by A2AR−/− macrophages engulfing apoptotic cells was inhibited by the adenylate cyclase activator forskolin (Fig. 6E), while it was enhanced in wild type macrophages by the protein kinase A inhibitor Rp-cAMP triethylamine (Fig.6F) suggesting that A2AR-mediated adenylate cyclase signaling inhibits primarily NO production.
NO can be produced by three nitric oxide synthetase isoenzymes, two of which, endothelial (e)NOS and inducible (i)NOS, are found in macrophages. Endothelial NOS is expressed constitutively, and its activity is dynamically regulated by Ca2+ - calmodulin. Inducible NOS is not expressed normally, and is highly inducible. The capacity of NOS to synthesize NO also is determined by the amount of its substrate, arginine, the level of which is negatively regulated by arginases (36). We investigated the expression of various genes encoding enzymes related to NO production, such as arginase I and II, iNOS and eNOS. eNOS was not detectable in resting or engulfing macrophages, suggesting that iNOS is responsible for the apoptotic cell-associated NO production. In line with higher NO production, the expression of iNOS was significantly higher in A2AR−/− macrophages than in their wild type counterparts (Fig. 7A). Exposure to apoptotic cells did not alter the levels of arginase I, but induced a downregulation in the arginase II expression, favoring the utilization of arginine in the production of NO. The downregulation of arginase II was more pronounced in A2AR−/− macrophages. In addition, the downregulation of iNOS by apoptotic cells was delayed in A2AR−/− macrophages (Fig. 7B). All these data indicate that exposure to apoptotic cells and loss of A2AR induces a modification in arginine metabolism that favors NO production.
Since the A2AR-induced adenylate cyclase pathway suppresses NO production, we tested whether influencing the adenylate cyclase pathway alters arginase II or iNOS expression. As shown in Figure 7C, inhibition of protein kinase A by Rp-cAMP triethylamine in wild type macrophages enhances the expression of iNOS, while the addition of forskolin to A2AR−/− macrophages inhibits it (Fig. 7D). Similar manipulations altered the expression of arginase II conversely.
Acute inflammation normally resolves by mechanisms which are initiated in the first few hours after an inflammatory response begins. The resolution program involves phagocytosis of apoptotic neutrophils by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines such as TGF-β1 (37).
The present study investigated whether adenosine and one of its receptors, the adenosine A2A receptor, could be involved in the anti-inflammatory response induced in macrophages by apoptotic cells. We have shown that macrophages engulfing apoptotic cells produce adenosine at levels that can trigger adenosine A2A receptors and, at the same time, elevate the expression of the receptor itself. Thus, adenosine can act in an autocrine manner during phagocytosis. Loss of adenosine A2A receptors did not affect the rate of phagocytosis. This was a surprise for us, as increases in cAMP levels were reported to inhibit engulfment of apoptotic cells (38). However, when exposed to apoptotic cells, A2AR−/− macrophages notably produced increased amounts of MIP-2 and KC acting as chemoattractants for various cell types, especially for neutrophils. We could confirm these data using a specific A2AR antagonist indicating that lack of actual A2AR signaling rather than altered macrophage differentiation in the absence of A2AR explains the phenomenon. These data indicate that during engulfment of apoptotic cells, especially when macrophages participate in the resolution of inflammation, where they clear large numbers of apoptotic neutrophils (39), A2ARs might participate in the negative feedback control of neutrophil transmigration to the inflammation site. Since inflammatory cytokines were shown to sensitize A2ARs (40), the role of adenosine mediating the inhibitory effect of apoptotic cells might be more significant under inflammatory conditions, than it was observed in our in vitro model, which lacked inflammation. In support of this hypothesis, enhanced production of MIP-2 and KC by A2AR−/− macrophages engulfing apoptotic cells was shown in an in vivo peritonitis model, and this was accompanied by MIP-2- and KC-dependent neutrophil migration.
In our further experiments, MIP-2 production by A2AR−/− macrophages was studied in details. Though previous studies have shown that apoptotic cell-induced IL-10 production in macrophages can negatively regulate the production of proinflammatory cytokines (4), and A2ARs were reported in certain inflammatory contexts to promote IL-10 formation (41), we found no detectable IL-10 production in our experimental system. Instead, we found that MIP-2 synthesis was partially related to an enhanced NO production by A2AR−/− macrophages engulfing apoptotic cells that regulated MIP-2 production on transcriptional level. Enhanced NO production of A2AR null macrophages as compared to the wild types might be related to higher levels of iNOS, which produces NO, and lower levels of arginase II, which normally degrades arginine, the substrate of NO synthesis. However, mRNA levels alone might not reflect the real activities or activity ratio of these enzymes, as just iNOS activity alone was shown to be regulated by various signals on transcriptional, mRNA, translational and posttranslational levels (42, 43). In support of our hypothesis, however, alterations in the arginine metabolism (favoring the arginase pathway leading to polyamine synthesis and inhibiting the synthesis of NO) following engulfment of apoptotic cells have already been reported (44). Interestingly, both TGF-β released by macrophages engulfing apoptotic cells (42, 45) and compounds known to activate protein kinase A (46, 47) were shown to increase arginase activity and decrease NO production in macrophages indicating that both TGF-β and adenosine A2A receptors, that activate protein kinase A, might mediate the effect of apoptotic cells on the arginine metabolism of engulfing macrophages. The role of TGF-β was proven previously (44), while our data indicate the additional involvement of A2ARs. All together our data demonstrate for the first time that besides TGFβ and IL-10 (4,5) adenosine also participates in the negative regulation of pro-inflammatory cytokine production of macrophages engulfing apoptotic cells. In this context adenosine uses the A2A receptor pathway and inhibits primarily neutrophil chemoattractant formation and the consequent neutrophil immigration.
The help of László Virág's lab in NO determinations and the excellent technical work of Edit Komóczy and Zsolt Hartman are gratefully acknowledged.
1This study was supported by Hungarian grants from the National Research Fund (K77587, TS-44798, F67632).
3Abbreviations used in this paper: A2AR, adenosine A2A receptor; KC, cytokine-induced neutrophil-attracting chemokine; L-NAME, L-(G)-Nitro-L-arginine methyl ester; LXR, liver X receptor, iNOS, inducible nitric oxide synthetase; IP-10, interferon-gamma inducible protein 10 kD; PPAR, peroxisome proliferators activated receptor; TGF-β, transforming growth factor-β