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The Journal of Immunology, 2007, 179, 1884 -1892
Copyright © 2007 by The American Association of Immunologists, Inc.
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Adenosine Deamination Sustains Dendritic Cell Activation in Inflammation1

Melanie D. Desrosiers*, Katherine M. Cembrola{dagger}, Michael J. Fakir*, Leslie A. Stephens*, Fatimina M. Jama*, Afshin Shameli*, Wajahat Z. Mehal{ddagger}, Pere Santamaria* and Yan Shi2,*

* Department of Microbiology and Infectious Diseases, Immunology Research Group and Julia McFarlane Diabetes Research Centre, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; {dagger} Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655; and {ddagger} Department of Internal Medicine, Yale University, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adenosine is a suppressive agent that protects the host from excessive tissue injury associated with strong inflammation. In tissue stress, higher levels of adenosine signal through adenosine receptors to exert strong anti-inflammatory effects, and thus protect host cells. Existing evidence also suggests that elevated adenosine potently down-regulates the activation of lymphocytes during inflammation. This notion, however, is in contrast with another basic observation that the immune system is highly activated precisely under the same circumstances against pathogens. In this study, we show that inflammatory responses of dendritic cells (DCs) are highly sensitive to adenosine suppression. However, they intrinsically carry high adenosine deaminase activity, which in turn degrades and removes adenosine from the surroundings, cutting off DCs from the suppression. This regulatory mechanism is important in DC responses to pathogen-associated molecular patterns and their activation of T cells. Our findings suggest a mechanism that DCs maintain their hyperreactive state in inflammation despite the general state of suppression, and reveal a regulatory role of adenosine deaminase in DC innate immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Inflammation is a vital component of host defense. Excessive inflammation, however, can cause tissue damage among other unfavorable effects. Adenosine has long been recognized as an important factor that negatively regulates inflammatory responses, and it serves as a nonredundant innate mechanism that protects host tissues in disease conditions (1, 2, 3). In tissue stress, such as ischemia and infection, extracellular adenosine levels rise and signal through G protein-coupled P1 adenosine receptors (P1Rs)3 on host cells (4, 5). This signaling event in general results in elevated cAMP activity that down-regulates host cell activation. The protective effect of adenosine is evident. In P1R knockout mice, inflammation leads to massive tissue damage (6). It is also well known that adenosine is an important factor of protection in ischemia and other aseptic tissue damage (7, 8, 9). This common catabolite has therefore been termed the retaliatory nucleoside, in reference to its active defense against cellular stress (2, 3).

The suppressive effect by adenosine regulates immune functions as well. In both mice and humans, adenosine signaling, particularly via P1 A2A and A2B receptors on macrophages, inhibits inflammatory cytokines while stimulating IL-10 production (10, 11). It also reduces TNF-{alpha} and increases IL-6 production of cord blood monocytes, which is suggested to confer the susceptibility to microbial infections in human newborns (12). However, in the context of adenosine-mediated immune suppression, one interesting observation is the APC activation in inflammation and infection, particularly their strong inflammatory responses toward pathogen-associated molecular patterns (PAMPs). This issue is important with regard to dendritic cells (DCs), which lie at the center of immune induction when the levels of extracellular adenosine are known to be the highest (13, 14, 15). Understanding mechanisms that mediate this resistance to adenosine suppression will reveal an important feature of DC biology.

Metabolically, adenosine deaminase (ADA) is the enzyme that catabolizes adenosine (16). ADA does not have its own hydrophobic/transmembrane domain, and is present both intracellularly and extracellularly. As expected, blocking extracellular ADA enhances adenosine signaling on T cells (17). On some human cells, independent of its enzymatic activity, extracellular ADA is associated with CD26 (18, 19, 20). The complex has been studied in great detail (18, 19). Engaging CD26 delivers a stimulatory signal to T cells via CD45 (21). Dong et al. (22) further demonstrated that in the presence of adenosine, a T cell line with surface CD26/ADA showed reduced IL-2 production and proliferation in response to CD3 or PMA stimulation. In vivo CD26/ADA complexes are not known to be present on the DC surface (20, 23). Furthermore, unlike in humans, rodent CD26 is not known to be associated with ADA due to sequence variations (24, 25, 26). The notion of CD26 signaling complexes, therefore, would fail to explain the strong resistance of DCs to adenosine suppression in inflammation. In contrast, ADA was detected on the surface of human APCs, most likely via alternative anchoring mechanisms, such as by interacting with A1 or A2B adenosine receptor (20, 27, 28). Using super-Ag stimulation, a recent study shows that ADA anchored in such a manner may interact with CD26 on T cells as part of immune synapse (20). However, how ADA affects DC activation and how DCs escape adenosine suppression remain unknown.

Our work on the question revealed a regulatory role for ADA on DCs that is directly tied to its enzymatic activity. We found that DCs were highly sensitive to adenosine inhibition. This inhibition was mediated via the A1 receptor. Importantly, the sustained activation of DCs in response to inflammatory stimuli was dependent on their ADA enzymatic activity. Our data show that DC ADA actively removed adenosine, potentially cutting off DCs from immune suppression. This regulatory mechanism reveals new insight into DC biology and provides a logical explanation as to how DCs remain highly responsive to PAMPs in the ubiquitous adenosine-mediated cell suppression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cells, and reagents

Female C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory and Charles River Laboratories, and used between 7 and 10 wk of age. Female NOD and 8.3 TCR transgenic NOD mice (5–10 wk of age) were maintained at the University of Calgary animal facilities. All mice were maintained in a specific pathogen-free facility at University of Calgary, according to the Institutional Animal Care and Use Committee guideline. THP-1 and DC2.4 cells were gifts from P. Cresswell of Yale University (New Haven, CT) and K. Rock of University of Massachusetts Medical School (Worcester, MA), respectively. Cell lines were routinely cultured in RPMI 1640 with 10% FBS plus 1 mM HEPES, 25 µM 2-ME, and pen/strep antibiotics. All reagents were purchased from Sigma-Aldrich, unless otherwise indicated. ADA Abs were purchased from Rockland (100-401-140) and Santa Cruz Biotechnology (H-300). Mouse CD26 Ab was purchased from Abcam (ab17538). A1R Ab was from ABR (PA1-047). A2AR (H-82), A2BR (N-19), and A3R (H-80) were purchased from Santa Cruz Biotechnology. Secondary Abs were purchased from Jackson ImmunoResearch Laboratories. All other Abs and reagents were from eBioscience. Killed Escherichia coli was produced by adding 250 µg/ml ampicillin at 16 and 20 h after the bacteria inoculation in Luria-Bertani broth. The culture was stopped at 24 h and the bacteria pellet was subjected to freeze-thaw twice. This pellet was tested to be sterile in mammalian cell culture. CpG oligonucleotide 1826 was obtained from Coley. Poly(IC) was purchased from Amersham.

C57BL/6 or BALB/c bone marrow (BM) DCs were produced, as previously described, with 3 ng/ml rGM-CSF and 3 ng/ml rIL-4 (29). In all BM DC assays, 6- or 7-day DC cultures were gently flushed twice with warm cell culture medium to remove any DCs in suspension. DCs produced in this manner are ~90% CD11c positive. Splenic DCs were isolated using CD11c Ab-conjugated beads (Miltenyi Biotec) and were washed and resuspended with cell culture medium before the assay. THP-1 DC morphological transformation was induced by adding 10 ng/ml PMA to the culture for 24 or 48 h, followed by washing with cell culture medium.

ADA inhibitors and P1R agonists and antagonists

ADA inhibitors and P1R agonists and antagonists were obtained from the following sources and used at doses established in previous reports (10, 17, 30, 31, 32). Adenosine, 5-(N-ethylcarboxamido) adenosine (NECA), 6-((4-nitrobenzyl)thio)-9-beta-D-ribofuranosylpurine (NBMPR), N6-cyclopentyladenosine (CPA) and 3-propyl-6-ethyl-5-(ethylthio)carbonyl)-2-phenyl-4-propyl-3-pyridine carboxylate were obtained from Sigma-Aldrich. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), CGS 21680, MeCCPA, MRS 1706, ZM 241385, and DPCPX were from Tocris Cookson. The 8(p-sulfophenyl)theophylline (8-PST) was obtained from A.G. Scientific. Tetrahydrouridine was obtained from Calbiochem. Most of these reagents were dissolved in DMSO, per manufacturers’ instructions. Deoxycoformycin (DCF; Nipent) is a gift from Mayne Pharma (Paramus, NJ), and was dissolved in water before the assays. All preparations were warmed to 37°C, and immediately added to assay wells to avoid crystallization/precipitation.

DC stimulation and ADA inhibition assays

Medium in DC cultures in 24- or 12-well plates was first swirled and removed. The warm cell culture medium was used to replenish the wells (2 ml each for 12-well plates, and 1 ml for 24-well plates). PAMPs and inhibitors, as described, were then added, and the plates were incubated for 6 or 24 h in a 37°C 5% CO2 incubator. Supernatant was collected at indicated time points, frozen, and assayed together with Ready-Set-Go kits from eBioscience.

DC migration assay was performed, as previously described (33). Briefly, 3 µl of Flurobrite (Polysciences) FITC beads (0.5-µm-diameter polystyrene) was mixed with 106 EL4 cells (freeze-thaw three times), and injected in a total volume of 50 µl with or without 1 µl of 10 mM DCF s.c. on the hind flank of C57BL/6 mice. Forty-eight hours later, poplineal lymph nodes were collected, digested with 1 mg/ml collagenase (type II) and 0.1 mg/ml DNase, and then passed through a nylon mesh. Cells were then stained for CD11c and analyzed by FACS. All flow cytometry assays were done with a BD FACScan (BD Biosciences). Data were analyzed with Flowjo (Tree Star).

P1R staining was performed on 6-day BM DCs. Adherent DCs were removed by scraping and stained for CD11c. Cells were then washed and incubated with Cytoperm solution (BD Biosciences), incubated with FcR blocker (clone 93), and further stained in Cytowash with P1R Abs, described in Materials and Methods. Cells were analyzed by FACS.

Apoptosis assay

C57BL/6 BM DC (day 6) were flushed with 1x PBS, and fresh warm culture medium was added to the plate. Cells were treated with either 10 µM EHNA or DCF with 5 µM adenosine. After 6 and 24 h, cells were stained for annexin V using a kit from R&D Systems (TA4619). Cells were washed and resuspended in 100 µl of annexin V reagent containing 1 µl of annexin V-biotin and propidium iodide. After 15 min, these cells were then washed and resuspended in 100 µL of 1x binding buffer containing 1 µl of strepavidin-FITC (eBioscience 11-4317-87) for 15 min.

Biochemical analyses

HPLC analysis was performed with a Shimadzu Prominence system with a CBM-20-A controller, an LC-20AB fluid pump, and an SPD-M20A diode array detector. For DC ADA activity assays, instead of measuring the catabolite production of inosine, we measured the decrease of adenosine with EZ-start software automated peak area analysis. This was because BM DCs and DC cell line cultures produced disproportionally lower inosine compared with other cell lines, suggesting purine nucleoside phosphorylase activity. Briefly, BM DCs or other control cells were scraped off culture plates with a rubber policeman, and washed once in PBS and twice in RPMI 1640 (no serum), and resuspended in 3 ml of RPMI 1640 at 106/ml. A total of 9 µl of 5 mM adenosine was added to the suspension. EHNA was added to the control setup to be used as the reference of remaining adenosine without ADA activity. NBMPR (10 nM) was added to block adenosine transportation across the plasma membrane. A total of 400 µl of samples was taken at times indicated and moved into vials with 1 µl of 1 mM EHNA (to stop the reaction). The samples were then spun for 2 min with a microcentrifuge, and 100 µl of the supernatant was injected into the HPLC. The samples were analyzed on a Restek C18 column with 0.1% trifluoroacetic acid as buffer A and 0.65% trifluoroacetic acid/80% acetonitrile as buffer B. Adenosine was resolved by a 0–15% B gradient at 1 ml/min. HPLC analyses with EZ-start software were done at UV 260 nM. Peak area analysis was performed with the default peak selection mode. The peak area as a percentage of total absorption at 260 nm was used for the calculation to minimize run to run or injection volume variability.

ADA-blocking and depletion assays

A total of 5 ml of protein A beads (RepliGen) was washed in PBS and incubated with 2 ml of Rockland ADA Ab, in pH 8.5 buffer for 20 min with tumbling. The Ab was then cross-linked to the matrix with freshly opened 20 mM dimethyl pimelimidate dry powder (Pierce), and the reaction was terminated by adding glycine to the mixture. FBS was then passed through a free flow column filled with the protein A/ADA Ab beads. The beads were then regenerated by acid wash (0.1 M glycine (pH 2.6)), and the FBS elute was further depleted one or more rounds. The depletion was confirmed by HPLC analysis of adenosine to inosine conversion.

Transwell assays were similar to other EHNA-blocking assays. Briefly, DCs in 12-well plates were flushed twice. Warm ADA-depleted culture medium was replenished to cover attached cells, alone or with 1 µM EHNA + 5 µM adenosine, as well as indicated PAMPs. Transwells (0.4-µm pore; BD Biosciences) with 100 µl of the protein A ADA beads were sat immediately above the DC monolayer. Cytokine production was analyzed by ELISA 24 h later.

The 8.3-CD8 and OT-1 T cell activation

NOD DCs were produced similarly as with other common laboratory strains. NOD or C57BL/6 DCs were flushed twice and replenished with fresh warm medium. They were then incubated with indicated treatments overnight, followed by a pulsing with 10–8 M of either NRP-V7 or a control TUM peptide (NOD DCs), or 10–10 M SIINFEKL (C57BL/6 DCs) for an additional 1 h. Cells were then scraped off the plate and washed twice. Ten thousand DCs were mixed with 100,000 splenocytes in a total volume of 200 µl in 96-well plates (or 100,000 DCs with 4 million T cells in 12-well plates). The mixture was incubated for 48 h for the NOD DC culture, or as indicated in the C57BL/6 DC culture. For the NOD DC assay, [3H]thymidine was added into the culture for the last 24 h. The plates were then read by scintillation counting (Wallac Micro-beta 1750).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Adenosine inhibits DC activation in responses to PAMPs

We first observed the suppressive effect by adenosine on DCs by accident. In our study of activation markers of DCs stimulated by TLR ligands, we noticed that a control agent, ADA inhibitor EHNA, blocked the expression of activation markers of DCs incubated with poly(IC) or CpG (Fig. 1A). The observation seemed to indicate that inhibition of extracellular ADA and a rise of adenosine blocked DC activation in response to PAMPs. Because NF-{kappa}B activation is a key event in innate responses, we studied the effect of EHNA and high extracellular adenosine on NF-{kappa}B activity in response to TLR ligands. We used a NF-{kappa}B promoter-linked luciferase reporter construct (34). Although TLR9-transfected HEK cells responded well to CpG stimulation in their NF-{kappa}B activation, it was significantly reduced by EHNA (Fig. 1B). Adenosine alone at a high concentration (100 µM) shut down the NF-{kappa}B activation as well. Conversely, tetrahydrouridine, a cytidine deaminase inhibitor, and nitrobenzylthioinosine (NBMPR), an inhibitor of cellular equilibrative transporter of nucleosides, did not show any differences. The ineffectiveness of the membrane transport blocker NBMPR suggested that EHNA was acting on an extracellular signaling event. The same effect was seen with poly(IC)-mediated TLR3 triggering as well (data not shown). Our finding argues for a NF-{kappa}B inhibition possibly mediated by adenosine (35, 36).


Figure 1
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  		FIGURE 1. Adenosine inhibits DC activation in responses to PAMPs. A, Left panel, 7-day C57BL/6 BM DCs were flushed twice with warm medium, and either left untreated (shaded area) or incubated with 5 µg/ml CpG with (heavy line) or without (thin line) 10 µM EHNA for 5 h. CD86 expression was determined by FACS. Right panel, Identical with the left except that 100 µg/ml poly(IC) was used in place of CpG. B, HEK 293 cells were transfected with mouse TLR9, and a luciferase reporter gene under a NF-{kappa}B promoter, as well as a control renilla construct in 96-well plates for 18 h. They were then treated with either 5 µg/ml CpG, or 1 ng/ml mouse rIL-1, in the presence of one of the following reagents: 5 µM chloroquine, 1 µM EHNA, 10 nM NBMPR, 10 µM tetrahydrouridine, and 100 µM adenosine. The assay was performed for 6 h. The luciferase reporter activity was measured using a Promega dual luciferase kit. The values were shown as: (luciferase reading/renilla reading)/(untreated luciferase reading/renilla reading) – 1. IL-1 signaling does not require endosomal acidification, and therefore is insensitive to chloroquine, as expected.

 
PAMP-mediated DC inflammatory response is inhibited by extracellular adenosine

Because of the role of adenosine in inflammation control, we sought to study in detail how adenosine regulates DC inflammatory responses. We cultured DCs with EHNA and adenosine, and added CpG oligos to the culture. CpG stimulated strong IL-12 production at both 6 and 24 h. However, in the presence of EHNA, the IL-12 production was severely inhibited (Fig. 2A). TNF-{alpha} showed a faster onset of production at 6 h, without any significant tapering off at 24 h (Fig. 2B). Similarly, at both time points, its production was reduced by the presence of adenosine (Fig. 2B). It is important to note that in all subsequent IL-12 ELISA described in this study, TNF-{alpha} was tested simultaneously and consistently showed an identical trend.


Figure 2
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  		FIGURE 2. PAMP-mediated DC inflammatory responses are inhibited by extracellular adenosine. A, Immature BM DCs from C57BL/6 mice were grown in 1 ml vol in 24-well plates and then treated with or without 5 µg/ml CpG, and with or without 10 µM EHNA plus 5 µM adenosine, as indicated, for 6 or 24 h. In this and subsequent assays, EHNA treatment was always accompanied by 5 µM adenosine. The IL-12 was determined by an ELISA kit. B, Identical with A, except the TNF-{alpha} was measured. C, In this assay, 5 µg/ml CpG, 100 µg/ml poly(IC), and 1 ng/ml LPS were delivered simultaneously to DC cultures. ELISA were performed as in previous panels. D, Identical with the other panels, except that 10 nl of wet killed bacteria pellet (0.01% v/v) was added to DC cultures in place of other PAMPs. NECA (10 µM) was used alone to mimic the effect of adenosine. The 8-PST (100 µM) was also used to block adenosine-mediated suppression.

 
We next expanded our study to include two more canonical PAMPs, LPS and poly(IC). Adenosine/EHNA reduced the IL-12 and TNF-{alpha} responses by DCs to LPS and poly(IC) (data not shown). The ability of adenosine to effectively block individual PAMPs does not necessarily indicate any biological importance. In real-life infection, the host is exposed to a variety of assaults from microbial products. We therefore first tested the effect of adenosine on immature DCs in response to a combination of multiple PAMPs (LPS, CpG, and poly(IC)), as shown in Fig. 2C. Again, adenosine/EHNA blocked the inflammatory stimulations of this mixture of PAMPs (TNF-{alpha} data not shown). We repeated the assay with killed E. coli (sterilized by ampicillin treatment), which we believe represents a more comprehensive and realistic challenge to DCs. Surprisingly, in the presence of the bacteria, the production of IL-12 (Fig. 2D) and TNF-{alpha} (data not shown) by DCs was still potently down-regulated by adenosine signaling, similar to the effect of NECA, an adenosine analog and P1R agonist.

In Fig. 2D, the inhibition by EHNA was reversed by the addition of 8-PST (nonselective P1R blocker), confirming that the effect was mediated via adenosine receptor(s). The result also showed that DCs were functional in the presence of EHNA. Because it was important to rule out nonspecific activity of the treatment, we analyzed the treated cells further. We also tested DCF, another commonly used specific ADA inhibitor, and saw the same result (Fig. 3A). DCs treated with 5-fold assay concentration of EHNA remained morphologically indistinguishable from untreated controls for the duration of our assays. We also stained EHNA- or DCF-treated DCs with annexin V and propidium iodide to detect any signs of cell death or apoptosis. After 6 (data not shown) and 24 h in the presence of the inhibitors, the treated and untreated also remained indistinguishable by FACS (Fig. 3B). Taken together, these results suggest that ADA blockage led to adenosine accumulation that inhibited DC inflammatory responses.


Figure 3
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  		FIGURE 3. DC suppression is due to specific ADA inhibition. A, BM DCs were treated as in Fig. 2D. DCF (0.1 µM) was used to replace EHNA. The 8-PST (100 µM) was used alone or in combination with DCF treatment. IL-12 production was measured after 24 h of incubation. B, BM DCs treated with EHNA (10 µM) or DCF (1 µM) in the presence of 5 µM adenosine were analyzed for their annexin V staining and propidium iodide uptake after 24 h, as described in Materials and Methods.

 
Innate responses of DCs are blocked by the signaling of adenosine receptor A1

To study whether any adenosine receptor(s) mediates this inhibition, we individually added a list of selective P1R subtype agonists to our DC stimulation culture. In place of adenosine/EHNA, an A1 receptor agonist, CPA, acted similarly in DCs’ response to CpG, reducing both IL-12 (Fig. 4A) and TNF-{alpha} (data not shown) production. Similar results were obtained in the response to killed E. coli (Fig. 4B) (TNF-{alpha} not shown). In this study, another A1 receptor agonist, MeCCPA, also blocked the bacteria-induced cytokine production. Therefore, in all of these assays, A1 receptor agonists behaved similarly to adenosine in mediating the suppression.


Figure 4
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  		FIGURE 4. Innate responses of DCs are blocked by A1 adenosine receptor signaling. A, DCs were treated with CpG plus one of the following P1R agonists: EHNA plus 5 µM adenosine, 10 µM CPA (A1 agonist), 1 µM CGS (A2A agonist), 100 nM IB-MECA (A3 agonist), or 10 µM NECA (nonselective agonist). No good specific A2B agonist is currently available commercially. B, Similar to C, except that 0.01% (v/v) wet killed E. coli (BAC) was used in place of CpG, plus the addition of another A1R agonist MeCCPA. IL-12 was measured after 6 and 24 h. C, DCs were treated with either 5 µg/ml CpG alone, or CpG and 1 µM EHNA plus 5 µM adenosine. All the rest of the bars were treated with CpG and EHNA plus adenosine, and with one of the indicated P1R antagonists as follows: 10 µM DPCPX (A1 antagonist), 10 nM ZM241385 (A2A antagonist), 5 µM MRS 1523 (A3 antagonist), 10 nM MRS 1706 (A2B antagonist), or 100 µM 8-PST. Culture supernatants were collected after 24 h and cytokines were measured by ELISA. D, BM DCs were stained for CD11c expression and permeabilized and stained for P1R expression. The histogram for CD11c-positive cells is shown. The shaded area is second Ab only.

 
We also performed P1R antagonist studies. Adenosine blocked the response as expected in CpG (Fig. 4C)- and killed E. coli (data not shown)-stimulated cytokine production by DCs. This inhibition was mostly reversed by 8-PST, as well as partially by DPCPX, a selective A1R blocker. ZM 241385 (A2AR blocker), MRS 1523 (A3R blocker), and MRS 1706 (A2BR blocker) showed no or little effect. These results again showed that the adenosine-mediated inhibition is most likely mediated by A1 adenosine receptor. The partial reversal of cytokine production with A1R and pan-P1R inhibitors could be due to the incomplete blocking of highly sensitive A1R.

To support the role of A1R in adenosine-mediated suppression of DCs, we stained BM DCs for their A1R, as well as other P1R expression. Clearly, A1R expression on DCs was detectable by FACS (Fig. 4D). A3R was marginally expressed, whereas both A2Rs were undetectable.

Mouse DCs possess strong ADA activity

Adenosine alone at the concentration used above (5 µM, at the lower end of in vivo inflammatory responses) mediated very little blocking of TLR-triggered cytokine production in the absence of EHNA (data not shown). The necessity of EHNA in seeing the effect on DCs suggested a role of ADA in removing adenosine in DC responses to PAMPs. ADA is a plasma component. Steady-state adenosine catabolism by plasma ADA is a basal factor impartial to any particular cell type. DCs are therefore expected to show an additive capacity to remove adenosine in comparison with control cells.

We used the HPLC UV scanning method to identify and analyze the catabolism of adenosine in the presence of DCs (Fig. 5A). We tested BM DCs and a control cell line for their abilities to remove adenosine, compared with samples with EHNA taken at the same time points. It is clear that BM DCs removed adenosine faster than a control fibroblast line (Fig. 5B). Adding adenosine transport blocker NBMPR to the mixture slightly slowed down the conversion, suggesting that intracellular ADA plays an appreciable role in removing extracellular adenosine.


Figure 5
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  		FIGURE 5. DCs possess surface ADA. A, Chromatographic analysis of adenosine deamination. BM DC or B16 cells were scraped, washed, and resuspended in RPMI 1640 (serum free). Their ADA activities were analyzed, as described in Materials and Methods. Samples were spun, and the supernatants were analyzed with diode array UV absorption scanning profile. The 5th to 19.5th min is shown. Adenosine peak is indicated at both 0 and 1 h. B, Adenosine peak area analysis at 262 nm was recorded as a percentage of the total 262 nm absorption for the whole profile (to minimize the run to run or injection variations). The value is compared with the same incubation in the presence of EHNA (no enzymatic conversion), and expressed as the percentage conversion of adenosine. C, Staining of BM DCs vs a human monocytic DC cell line THP-1 for the expression of surface ADA, with two Abs. Heavy line: an Ab raised against human ADA (Santa Cruz Biotechnology), cross-reactive to mouse ADA, and the thin line: an ADA Ab raised against bovine ADA (Rockland). D, C57BL/6 lymph nodes were collected from multiple locations and pooled. Single-cell suspension was produced by frosted micro slide grinding. The cells were blocked with 2.4G1 (BD), and then stained with ADA Ab (FITC) plus one of the following PerCP Ab: anti-CD11c (eBioscience 35-0114), anti-CD4 (BD L3T4), anti-CD8 (BD LY2), or anti-B220 (BD RA3). ADA expression on various cell types was then measured by FACS. Each bottom histogram is the same as the P1 gate defined in the dot plot immediately above.

 
ADA secretion is a documented event, although the exact mechanism is still not clear. The ADA activity unaffected by NBMPR might have come from endogenous ADA that was being actively secreted. Our observation, however, could also indicate extracellular ADA attached on the membrane of mouse DCs. Although mouse CD26 and ADA do not form complex, other surface ADA-anchoring mechanisms in humans, such as A1 and A2B receptors, have been suggested. We therefore stained BM DCs with two ADA-specific Abs. Surprisingly, we were able to detect an epitope on mouse DCs, although the staining intensity was lower than that of a human DC line (THP-1). This epitope was seen on splenic DCs and B cells as well, and absent from resting primary CD4 and CD8 T cells. Whether this epitope was associated with the membrane ADA activity is unknown.

DC-intrinsic ADA sustains their response to PAMPs

A critical experiment that could demonstrate the unique resistance to adenosine was to show that blocking of DC ADA reduced their inflammatory response. Commercial sera have ADA activity that converted adenosine to inosine (Fig. 6A), as shown by HPLC analysis. The catabolite collected from HPLC was further analyzed by electro spray ion trap mass spectrometry, and showed a –267 ion in the negative ion mode, also identical with inosine (data not shown). We removed ADA in cell culture serum by multiple rounds of immune depletion with ADA Ab conjugated to a protein A column. The serum showed no detectable ADA activity after the treatment (Fig. 6A). With this serum medium, DC inflammatory responses to PAMPs were blocked by the addition of EHNA (data not shown), suggesting the DC-intrinsic ADA mediated the resistance to adenosine suppression. In our assay settings, it was possible that small quantities of free ADA come from cultured cells and may accumulate over the course of the assay. To deal with this possibility, we cultured DCs in Transwell plates. We added the protein A ADA Ab beads into the Transwells. Because the permeable bottom of the Transwells sat immediately above the DC layer, secreted free ADA would be removed by the Ab. With this setting, TNF-{alpha} and IL-12 production in response to PAMPs was still significantly blocked by the EHNA inhibition, very similar to assays with ADA-rich serum medium (Fig. 6B). Therefore, the result suggests that DC-intrinsic ADA sufficiently maintains DC activation in responses to PAMPs by removing extracellular adenosine, whereas free serum ADA was not necessary.


Figure 6
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  		FIGURE 6. DC membrane-proximal adenosine deamination. A, The protein A ADA column was produced, as described in Materials and Methods. A total of 20 µl of 5 µM adenosine was added to 1 ml of PBS with 10% of regular FBS or the ADA-depleted FBS. Thirty minutes later, the reaction was stopped by adding 1 µM EHNA, and 50 µl of the samples was analyzed by HPLC. Top panel, Regular FBS; bottom panel, ADA-depleted FBS. Spectra from the 9th to the 13th min are shown. B, BM DCs were assayed in ADA-depleted cell culture medium, with the addition of indicated PAMPs and with or without EHNA + 5 µM adenosine. A Transwell with 100 µl of protein A/ADA Ab was placed immediately above the DC monolayer. ELISA was performed after 24 h of culture. Similar settings without the Transwells produced similar data (data not shown).

 
Adenosine regulation on DC Ag presentation

We further evaluated the role of adenosine deamination in primary DC activation. In settings identical with BM DCs, splenic DC responses to PAMPs were equally suppressed by EHNA treatment (Fig. 7A). We further analyzed the DC inflammatory responses using a technique established in our laboratory. Endogenous DCs phagocytize particulate Ags in the periphery and move to local draining lymph nodes. This measurement of DC activation is significantly facilitated by local cell injury (mimicked by injecting freeze-thaw killed syngenic cells) (33). When we coinjected fluorescence-labeled beads with killed EL4 cells into the hind flanks of B6 mice, an addition of DCF completely prevented the DC migration (Fig. 7B). This result suggests that ADA inhibition blocked endogenous DC inflammatory responses as well.


Figure 7
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  		FIGURE 7. Adenosine regulation on DC Ag presentation. A, Primary splenic DCs isolated from C57BL/6 mice (see Materials and Methods) were assayed as in Fig. 2A. Fifty thousand splenic DCs were cultured in 2 ml of medium with indicated treatments. TNF-{alpha} was assayed by ELISA after 24 h. In this setting, primary DCs produced very little IL-12. B, FITC-labeled polystyrene beads were mixed with 106 killed syngenic EL4 cells, with or without DCF. The mixture was injected s.c. into the hind flank of B6 mice. Forty-eight hours later, poplineal lymph node cells were harvested and gated on CD11c, and their FITC-positive cells were analyzed by FACS. A total of 25,000 CD11c+ cells is displayed, and the frequency of bead-containing/FITC+ cells is indicated. C, C57BL/6 DCs were incubated with 5 µg/ml CpG alone or with EHNA plus adenosine overnight. In the last hour, some cells were pulsed with 10–10 M SIINFEKL peptide for 1 h, as indicated. They were then washed and mixed with OT-1 splenic cells. IFN-{gamma} production was measured at time points indicated. D, Identical with B, except that NOD DCs and NPR-V7 peptide were used. They were then washed and mixed with 8.3 NOD splenic cells. [3H]Thymidine was added 48 h after the culture and further cultured for 24 h. The 3H uptake was analyzed by scintillation counting.

 
We then performed assays to determine whether the loss of ADA function on DCs led to decreased Ag presentation by the treated cells. This was investigated by assessing the ability of EHNA to inhibit the presentation of OVA epitope peptide (SIINFEKL) by C57BL/6 DCs to OT-1 T cells in the presence of CpG. The addition of EHNA nearly completely blocked the OT-1 IFN-{gamma} production at multiple time points measured (Fig. 7C). This is consistent with the reduced CD86 expression shown in Fig. 1. We extended the assay by studying the Ag presentation of a naturally occurring endogenous T cell epitope (NRP-V7, mimotope for residues 206–214 of islet-specific glucose 6 phosphatase catalytic subunit-related protein) to autoreactive T cells (37, 38, 39). We pulsed DCs with NRP-V7 peptide in the presence of EHNA. Pulsed cells were then used to stimulate splenic cells from 8.3-TCR transgenic NOD mice. The 8.3-CD8 T cells are specific for IGRP206–214 in the context of Kd. Clearly, EHNA-treated DCs again demonstrated a reduced ability to activate Ag-specific T cells (Fig. 7C). These results suggest that EHNA or high adenosine levels inhibited the presentation of MHC class I-restricted Ags by DCs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The current consensus of adenosine signaling on APCs is as follows: rising adenosine levels prevent the overreaction of these important immune cells, and therefore limit their ability to activate other cells. In LPS-stimulated human monocytes or monocytic cell lines, the production of TNF-{alpha}, IL-6, IL-8, and IL-12 was reduced by high adenosine. The effect was mediated by A2R, and often accompanied by a concomitant rise of IL-10 (2, 10, 11, 31, 40, 41). In human DCs, high adenosine also suppressed IL-12 and TNF-{alpha} production (42). Adenosine as an innate signal therefore has been proposed as the retaliatory nucleoside that helps the host suppress overreactivity, particularly that of immune cells (2, 3). However, in cases of tissue distress, the immune system is almost inevitably activated, in response to either exogenous microbial assault or internal cell injury (43, 44). Our data suggest that to DCs, adenosine-mediated negative regulations may be overcome by their strong ADA activity.

Adenosine signaling on DCs under inflammatory conditions is currently understudied. We therefore performed a list of assays that studied the premise of DC activation in the presence of adenosine. We found the following: 1) DCs are sensitive to even modest amounts of adenosine under ADA blockage; 2) this suppression is mediated by A1R; 3) DCs possess strong ADA activity; and 4) the DC-intrinsic ADA is sufficient and necessary to fend off adenosine-mediated suppression. The last point may explain the robust activation of DCs and the immune system in the presence of strong adenosine suppression in disease conditions.

Accommodating the general consensus on adenosine suppression, our work seems to indicate that in tissue stress, the equilibrium of adenosine reached by the balance of adenosine production and serum ADA is high enough to protect most host tissues. At the same time, strong ADA on DCs lowers adenosine levels in the surrounding and therefore avoids the inhibition.

ADA plays a crucial role in immune cytogeny. Its deficiency leads to human SCID and profound immune deficiency in mice (45, 46, 47). It is interesting that after the immune system development, ADA remains the highest in the lymphoid tissues (48, 49, 50), which suggests biological function. The significance of surface ADA on T cells has been extensively studied (18, 19, 20). In our study, the strong ADA activity in DCs also fits into the general scheme of immune activation. In infection, APCs are the first to be activated in vivo because of their extraordinary abilities to sense PAMPs. The high ADA activity on APCs in resting lymph nodes supports this line of reasoning because it will make APCs insensitive to the initial surge of adenosine at the time of infection or tissue stress.

It is likely that surface-attached ADA detected in our assays converted adenosine, reducing the inhibition via P1Rs in their responses to PAMPs. This mode of action would fit the general understanding of adenosine signaling in immune cells. Clearly, intracellular ADA contributes to the regulation of extracellular adenosine as well, most likely via the cell membrane transporter. Alternatively, one possibility is that DCs actively secrete the enzyme to convert exogenous adenosine. The secretion has been reported in T cells (22). Lastly, it is still possible that intracellular ADA directly regulates a signaling event inside the cells, although it would be more difficult to understand in light of the fact that P1R blocking by 8-PST and A1R antagonists strongly reversed the inhibition, indicating a surface receptor activity. One should be reminded that on human DCs, extracellular ADA expression is documented, implying that surface ADA-mediated adenosine reduction may impact human DC activation.

The potential CD26/ADA signaling complex on human T cells (18, 19, 21) is not to be expected because of protein sequence differences. One mouse DC line, DC2.4, possesses strong ADA activity (similar to BM DCs), yet is CD26 negative (our own observation). The question remains whether the ADA activity detected on DC membrane is attached via any specific interaction. In humans, ADA can be attached to DCs via A1 and A2B receptors (20, 27, 28). Whether this attachment is seen in mice has not been reported. Interestingly, BM DCs were stained positive with two of our ADA Abs (Rockland and Santa Cruz Biotechnology). This epitope was seen on mouse monocytic cells, splenic DCs, but not on T cells or B cells (our own observation). Whether the epitope is related to the enzymatic activity is unknown, because ADA Ab variability has been reported (51). More vigorous tests are needed. Unfortunately, the presence of the epitope may mask the real association of ADA and mouse DC surface for identification purposes. We are planning to use genetic approaches to study the issue.

In this study, an unexpected result was that A1R mediated adenosine suppression on DCs, a finding that is in contrast to previous reports in which A2AR and A2BR were often the transducer. P1R signaling is very complex. The high-affinity A1R has been regarded as a potential activator of immature DCs that senses low levels of adenosine in resting tissues (3). Because in our assays we continuously blocked ADA activity, adenosine accumulating at the surface of DCs could be higher than those in some other reports. The A1R may behave differently at the continuous stimulation of higher concentrations of adenosine. A potentially similar scenario has been seen in the pulmonary inflammation in ADA knockout mice in which mouse A1R showed an anti-inflammatory and protective role (52). It is interesting to note that genetic studies of mouse A1R have shown protective effects elsewhere as well, such as in ischemia injury and experimental allergic encephalomyelitis (53, 54).

Significant work remains to be done to fully understand the functions of DC-associated ADA. Ideally, biological functions of ADA on DC surface should be analyzed with ADA knockout mice and adoptive transfer experiments. A caveat is that serum ADA may be the source of the cell surface expression (22). If our observed ADA binding on mouse DCs was mediated by an association between a specific cell membrane entity and serum ADA, it would reduce the value of in vivo adoptive transfer experiments. The most definitive tool will come with a mouse model that completely lacks DC-associated ADA activity, a challenging proposal to the field because cell surface ADA anchoring is not very well understood.

Finally, we believe that surface ADA-mediated adenosine removal surrounding DCs may be common in immune inductions because the rise of adenosine in diseases and injuries is nearly an invariable event and DCs are at the center of many immune responses. It provides a regulatory mechanism in DCs’ innate response to PAMPs. It is perceivable that with ADA, enhanced activation of DCs in pathogen invasion is advantageous to the host. However, it could also be involved in autoimmunity, particularly in cases in which inflammation is a necessary component. Our findings therefore may have implications in immune regulation, vaccine development, and autoimmunity intervention.


   Acknowledgments
 
We thank Dr. Yang Yang for his input and discussion, as well as his gift of purified primary DCs; Dr. Kenneth Rock for this facility support; University of Calgary Immunology Research Group Flow Cytometry Core and Southern Alberta Mass Spec Centre for their technical support; Dr. Giuseppina Colarusso for her time and advice on imaging techniques; and Dr. Kate Fitzgerald and Mayne Pharma for reagents.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by a grant from National Institutes of Health (to Y.S.) A.S. is supported by a Ph.D scholarship from Alberta Heritage Foundation for Medical Research. Back

2 Address correspondence and reprint requests to Dr. Yan Shi, B872 HSC, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail address: yshi{at}ucalgary.ca Back

3 Abbreviations used in this paper: P1R, P1 adenosine receptor; 8-PST, 8(p-sulfophenyl)theophylline; ADA, adenosine deaminase; BM, bone marrow; CPA, N6-cyclopentaladenosine; DC, dendritic cell; DCF, deoxycoformycin; EHNA, erythro-9-(2-hydroxy-3-nonyl) adenine; NBMPR, 6-((4-nitrobenzyl)thio)-9-beta-D-ribofuranosylpurine; NECA, 5-(N-ethylcarboxamido) adenosine; PAMP, pathogen-associated molecular pattern. Back

Received for publication March 16, 2007. Accepted for publication June 9, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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