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Originally published online as doi:10.1189/jlb.0108046 on December 26, 2008

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(Journal of Leukocyte Biology. 2009;85:452-461.)
© 2009 by Society for Leukocyte Biology

Adenosine and IFN-{alpha} synergistically increase IFN-{gamma} production of human NK cells

Florian Jeffe1, Kerstin A. Stegmann1, Felix Broelsch, Michael P. Manns, Markus Cornberg and Heiner Wedemeyer2

Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany

2 Correspondence: Abteilung für Gastroenterologie, Hepatologie und Endokrinologie, Medizinische Hochschule Hannover, Carl Neuberg-Str.1, 30625 Hannover, Germany. E-mail: wedemeyer.heiner{at}mh-hannover.de


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ABSTRACT
 
Prevention of overwhelming immune reactions is essential for an organism to survive. Adenosine, a ribonucleoside produced by various cell types during inflammatory processes, has been shown to inhibit effector functions of different immune cells. Here, we show that the adenosine A3 receptor agonist iodobenzyl methylcarboxamidoadenosine potently inhibited proliferation, IFN-{gamma} production, and cytotoxicity of activated human lymphoid cells. Stimulation of the A3 receptor also caused apoptosis of activated PBMC. However, when PBMC were stimulated with IFN-{alpha}, adenosine did not decrease, but synergistically increased, the IFN-{gamma} production of NK cells. This effect was also mediated mainly via the A3 receptor. Thus, our data suggest that adenosine differentially contributes to the regulation of immune responses during inflammatory processes: It may increase effector functions of NK cells in combination with IFN-{alpha} but also prevents overwhelming immune responses by inhibiting proliferation and induction of apoptosis of activated lymphoid cells. Future studies need to define the role of the different adenosine receptors in more detail.

Key Words: IB-Meca • A3 receptor


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INTRODUCTION
 
Prevention of overwhelming immune reactions is essential for an organism to survive. Different mechanisms leading to an inhibition of immune responses have been described. One important molecule involved in the regulation of the immune network is adenosine [1 ].

Adenosine, an endogenous purine nucleoside, can have multiple functions in various tissues [2 ]. The extracellular adenosine concentration has been reported to rise as a result of nonlytic secretion of adenosine during hypoxic conditions and inflammatory processes [3 ], as well as in tumor environments [4 ]. In addition, adenosine can be generated extracellulary from ATP, ADP, and AMP by 5'-nucleotidase and -ectoapyrase in the brain and lung with a half-time of ~200 ms [1 ]. There is also a significant, nonlytic secretion of 5'-AMP by different cells, including neutrophils and mast cells, which is then converted to adenosine by the ecto-5'-nucleotidase (CD73) [5 ].

Adenosine affects multiple physiological functions in the nervous and cardiovascular systems. Its influence on the heart has been described already, almost 80 years ago. Adenosine is used therapeutically in the treatment of certain heart-rhythm disturbances [6 ]. It also plays an important role in the central and peripheral nervous system, including CNS development and regeneration [7 , 8 ]. Moreover, it is well known that adenosine is a potent regulator of the immune system, mainly by displaying an immunosuppressive function [9 , 10 ]. The lack of adenosine desaminase, an enzyme that is degrading adenosine, leads to a severe immunodeficiency [11 , 12 ]. Subsequently, gene therapy, introducing adenosine desaminase in hematopoetic stem cells, has been applied to patients with hereditary adenosine deaminase (ADA) deficiency 15 years ago [13 ].

Inhibitory effects of adenosine have been described for activated T cells [14 15 16 ], dendritic cells (DC) [17 , 18 ], NK cells [19 ], eosinophils, monocytes, macrophages, neutrophils, and mast cells [20 21 22 23 ]. Although in NK cells and T cells, immunosuppressive effects are, to a large extent, mediated via A2A/A2B receptors [24 , 25 ], inhibitory effects may also be mediated via the adenosine receptor A3 [26 ], one of four known G protein-coupled receptors (A1, A2A, A2B, and A3 receptor). However, subsequently, A3 agonists have been shown to inhibit degranulation of human eosinophils and neutrophils [27 28 29 ], chemotaxis and IL-12 production of DC [18 , 30 ], and release of proinflammatory cytokines by monocytes [31 32 33 34 ] and to induce apoptosis of PBMC [35 , 36 ].

The aim of the present study was to investigate in more detail the role of different adenosine receptors in regulating functions of mitogen-activated PBMC. Moreover, we were specifically interested whether adenosine also affected activation of PBMC by type I IFNs. Our interest in type I IFNs is based on its use in the treatment of viral hepatitis [37 ], showing some efficacy in inhibiting viral replication. However, about half of the patients with chronic hepatitis C virus (HCV) and two-thirds of patients with chronic HBV do not respond to IFN-{alpha} therapy [38 39 40 41 ], and thus, there is an urgent need to further improve the efficacy of currently available therapies. We have shown previously that HCV-specific T cell responses are impaired in chronic HCV [42 ]. The initial hypothesis for this study was that adenosine might inhibit activation of immune cells by IFN-{alpha} and thereby, be a potential target for therapeutic interventions. Indeed, we could show that adenosine potently inhibited different effector functions of activated human lymphoid cells and induced apoptosis via the A3 receptor. However, and to our surprise, IFN-{alpha} and adenosine did not decrease but increased IFN-{gamma} production of NK cells. Thus, our data suggest that adenosine differentially contributes to the regulation of immune responses during inflammatory processes by increasing effector functions of NK cells in combination with IFN-{alpha} but also by preventing overwhelming immune responses through inhibition of proliferation and induction of apoptosis of activated lymphoid cells.


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MATERIALS AND METHODS
 
Isolation of PBMC
Cells were isolated from healthy donors and from patients with compensated liver disease [viral HBV and/or HCV and primary biliary cirrhosis (PBC)]. Liver disease patients were recruited in the Outpatient Clinic of Hannover Medical School (Germany). All volunteers participated in this study after obtaining informed consent in a protocol that was reviewed and approved by the Ethics Committee, Hannover Medical School. Characteristics of the patients are indicated in the respective figure legends.

PBMC were isolated using density gradient centrifugation with Ficoll (Seromed, Berlin, Germany), as described before (Wiegand, Hepatology 2004). For all experiments, only fresh cells and never cryopreserved or pooled PBMC were used.

Proliferation of a PBMC culture
After separation of PBMC, the cells were resuspended in 10% AB medium [RPMI-1640 medium with 100 U/l penicillin, 100 µg/ml streptomycin, and 10% human AB serum (received from the blood bank of the Medical School of Hanover)]. Cells (200,000) were incubated on a 48-well flat-bottom plate (Nalge Nunc International, Denmark) in 500 µl medium, with or without different concentrations of adenosine (9-β-D-ribofuranosyladenine), coformycine [an inactivator of the adenosinedesaminase (Calbiochem, La Jolla, CA, USA)], 2-chloro-N6-cyclopentyladenosine (CCPA; Sigma Chemical Co., St. Louis, MO, USA), a highly selective adenosine A1 receptor agonist [inhibitor constant (Ki): A1=0.4 nM; A2=3900 nM], CGS-21680 hydrochloride (CGS; Sigma Chemical Co.), adenosine A2 receptor agonist (Ki: A1=2600 nM; A2=15 nM), iodobenzyl methylcarboxamidoadenosine (IB-Meca; Sigma Chemical Co.), a selective adenosine A3 receptor agonist (Ki: A3=1.1 nM) [43 ], PHA-M (Calbiochem), and several antigens. The plate was incubated at a temperature of 37°C with 5% CO2.

Concentration of adenosine and its agonists
In all experiments, we used concentrations of 2–50 µM adenosine and when needed, 0.2–20 µM specific agonists.

Antibodies
An IFN-{gamma} antibody (anti-human IFN-{gamma} mAb) and a biotin-labeled IFN-{gamma} antibody (anti-human IFN-{gamma} biotin-labeled mAb, purchased from Endogen, Woburn, MA, USA) were used for the ELISPOT assay. CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to isolate CD14+ cells from PBMC.

CFSE staining
The cells were suspended in 1 ml PBS and incubated for exactly 10 min with 2 µM CFSE. To stop CFSE uptake, cells were incubated with 100% FCS and then washed three times. Cells were incubated for 4–6 days before cell division was investigated by flow cytometry using a FACScalibur (Becton Dickinson, Heidelberg, Germany). Data were analyzed using the program Cellquest (Becton Dickinson).

ELISPOT assay
IFN-{gamma} ELISPOT assays were performed exactly as described using fresh PBMC only [44 ]. Calculation of responses was performed by an automatic ELISPOT counter, software A.EL.VIS (spot parameter, region of interest 80%; minimum size=25; minimum intensity=5; lamp brightness, 100%). Whenever the background in the medium was higher than 10 spots, we discarded the assay and did not make use of the results.

Intracellular cytokine staining
Fresh PBMC were separated from whole blood. Cells (500,000) in 10% AB medium were disposed in the wells of a 96-well round-bottom plate (Nalge Nunc International). Adenosine, IB-Meca, and IFN-{alpha} were added in the appropriate concentrations to a total volume of 100 µl. PMA/ionomycin was used as a positive control. The plate was incubated for 6 h at 37°C. After 1 h, Brefeldin A was added to a final concentration of 2 µg/ml. After incubation, the wells were washed twice with FACS buffer. The surface antibodies (CD14, CD56, CD3, CD107a) were added and incubated for 15 min at 4°C. After washing the plate once, 100 µl Cytofix buffer (Becton Dickinson) was added into each well. After 20 min at 4°C, the plate was washed with 200 µl Cytoperm/Cytowash (Becton Dickinson). Afterwards, FcR-blocking reagent was added for 15 min at 4°C. Finally, the plate was washed, and the intracellular antibody for IFN-{gamma} or TNF-{alpha} staining was added for 30 min at 4°C.

Cytotoxicity assay with 51Chromium release
PBMC were cultured for 10 days with IL-2 (20 U/ml). The day before the assay, the targets were prepared. JY-EBV-transformed cells (HLA-A2- and HLA-B2-positive) were cultured at 37°C and 5% CO2 overnight.

The next day, the different cell groups were incubated with 100 µCi 51Chromium and washed four times with HBSS. Meanwhile, we prepared the effector cells from fresh blood. We calculated with 3000 targets and E:T ratios (see Go Go Fig. 3B ).


Figure 1
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  		Figure 1. Adenosine inhibits IFN-{gamma} production and expansion of PHA-activated cells. (A and B) IFN-{gamma} production was measured via ELISPOT assay after incubation of 200,000 cells from healthy donors (not pooled) for 24 h. (C) PBMC (200,000) were incubated for 4 days with PHA (1 µg/ml), adenosine (Ade), and coformycin (Cof) at the indicated doses. Living cells were counted by excluding dead cells with trypan-blue staining. In both experiments, coformycin alone had no effect compared with the medium control (data not shown). For more information, see Materials and Methods. Experiments were repeated at least twice with comparable results.


Figure 2
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  		Figure 2. IFN-{alpha} does not alter the proapoptotic and antiproliferative effect of adenosine. (A and B) PBMC from healthy donors (not pooled) were incubated at the indicated conditions for 24 h. An Annexin/propidium iodide (PI) staining was performed and analyzed via flow cytometry. Again, coformycin alone had no effect compared with the medium control. (C) Cell counts over 4 days are shown. PHA concentration was 1 µg/ml.


Figure 3
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  		Figure 3. Adenosine A3 agonist inhibits proliferation of PHA-activated cells. (A) PBMC from healthy donors (not pooled) were stained with CFSE and incubated for 6 days with PHA and one of the three adenosine receptor agonists. On Day 6, cells were analyzed by flow cytometry. SSC-H, Side-scatter-height. (B) PBMC (200,000) were incubated for 4 days with SEB (1 µg/ml), IB-Meca, and MRS 1191 at the indicated doses. Living cells were counted by excluding dead cells with trypan blue staining.

Adenosine agonists were added during the cytotoxicity assay. We incubated the plates for 4 h after a short centrifugation at 37°C and 5% CO2. Spontaneous release was calculated with medium and maximum release with 10% Triton X-100 (Sigma Chemie, Deisenhofen, Germany). The supernatant (50 µl) was diluted with a 200-µl Szintillator (Optiphase Supermix, Wallac, Turku, Finland). The samples were analyzed with a β-counter (Microbeta 1450, Wallac).


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RESULTS
 
Adenosine inhibits IFN-{gamma} production of mitogen-activated PBMC
We first investigated the effect of adenosine on IFN-{gamma} production of PHA-stimulated PBMC. As shown in Figure 1 A and B , adenosine suppressed IFN-{gamma} production in a dose-dependent manner, as investigated by ELISPOT assays. Although 2 µM adenosine had almost no effect on IFN-{gamma} production, 10 µM adenosine caused a small reduction in spot-forming units (SFUs), and 50 µM adenosine induced a decline of IFN-{gamma} spots by more than two-thirds (Fig. 1A) . Coformycin has been shown to inhibit the endogenous ADA activity presented on the surface of activated T cells [45 ]. In line with these findings, coformycin was also required for adenosine to display its inhibitory function on IFN-{gamma} production of PHA-activated PBMC in our study (data not shown). Coformycin alone never had any significant effect on IFN-{gamma} production in any experiment.

Adenosine also inhibits the expansion of PHA-activated PBMC
As shown in Figure 1C , the number of living cells after 4 days of in vitro stimulation was reduced significantly by adenosine. This effect was only evident if coformycin were present. The inhibitory effect of adenosine was dose-dependent, as adenosine concentrations of 2 µM had almost no effect on cell numbers, and 50 µM adenosine inhibited cell proliferation completely and even reduced the number of life cells (data not shown).

Adenosine induces apoptosis of PBMC, which is not altered significantly by IFN-{alpha}
Adenosine causes cell death of PHA-stimulated PBMC [35 , 36 ]. However, it is unknown whether adenosine also causes cell death in PBMC stimulated with type I IFNs. We therefore studied the effect of adenosine on Annexin V expression by flow cytometry after 24 h of stimulation with adenosine and/or IFN-{alpha}. As shown in Figure 2A , 10,000 U/ml IFN-{alpha} alone had almost no effect on Annexin V expression as compared with the medium control. In contrast and as expected, 50 µM adenosine showed a significant increase of Annexin-V-positive cells. IFN-{alpha} and adenosine together showed only a minor, further increase of Annexin-V-positive cells, which was not statistically significant (Fig. 2B) . Adenosine was also more potent than IFN-{alpha} in inhibiting expansion of PHA-stimulated PBMC after 4 days (Fig. 2C) , as we also observed for Staphylococcal enterotoxin B (SEB)-stimulated cells (data not shown).

The inhibitory effect of adenosine on PHA-stimulated PBMC is mediated via the A3 receptor
The regulatory actions of adenosine have been shown to be mediated via four subtypes of adenosine receptors (A1, A2A, A2b, and A3) [2 ]. Therefore, we next investigated which receptor was responsible for the inhibition of PHA-stimulated PBMC by adenosine. PHA-stimulated PBMC were cocultured with adenosine receptor agonists CCPA (A1 receptor agonist), CGS (A2B receptor agonist), and IB-Meca (A3 receptor agonist). Cell proliferation was investigated on a single-cell level by CFSE stainings (Fig. 3A ). The percentage of living cells, as determined by forward-scatter and side-scatter characteristics after 6 days of in vitro culture, was not affected by CCPA and CGS coculture, and IB-Meca caused a significant reduction of cells in the living cell gate (7.2% vs. 41% for cells stimulated with PHA alone).

As expected, more than 80% of PBMC had reduced fluorescence signals for CFSE after PHA stimulation, indicating cell proliferation that was not influenced by CCPA and CGS. In contrast, almost no cell had divided after PHA stimulation if IB-Meca were present (Fig. 3A) . Thus, these data indicate that adenosine mediates its inhibitory effect on cell proliferation via the A3 receptor but may also cause cell death of PHA-activated PBMC.

As shown in Figure 3B , MRS 1191, a selective A3 receptor antagonist, counteracted the inhibitory effect of IB-Meca, supporting our theory that IB-Meca acts via the A3 receptor.

Stimulation of the adenosine A3 receptor inhibits cytotoxicity of PBMC
IB-Meca not only inhibited proliferation and IFN-{gamma} production of PHA-stimulated cells but also affected the cytotoxic activity of cultured PBMC, which were cultured in the presence of IL-2 for 10 days, and the cytotoxic activity was investigated by 51Chromium release assays. As shown in Figure 4 , IB-Meca but not CCPA and CGS abolished the cytotoxic activity of the PBMC almost completely. We did not investigate to what extent NK cells or T cells contributed to cytotoxicity of cultured PBMC.


Figure 4
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  		Figure 4. Adenosine A3 agonist inhibits cytotoxic activity of cultured PBMC, which were stimulated for 10 days with IL-2 (20 U/ml). Cytotoxic activity against the EBV-transformed B cell JY was analyzed via 51Chromium release assay after 24 h of incubation. For more information, see Materials and Methods. Experiments were repeated at least twice with comparable results.

Adenosine and IFN-{alpha} synergistically increase the IFN-{gamma} production of PBMC
We then were interested in whether adenosine also inhibits the stimulatory effect of IFN-{alpha} on PBMC. Stimulation of PBMC with IFN-{alpha} alone induced four to 14 (mean 8.3±3715) IFN-{gamma} spots per 2 x 105 PBMC. IFN-{alpha} concentrations of 1000–10,000 U/ml were required for significant numbers of IFN-{gamma} spots in the ELISPOT assay. Surprisingly, adding adenosine did not inhibit IFN-{gamma} production of IFN-{alpha}-stimulated PBMC but induced significantly more IFN-{gamma} spots (Fig. 5A ).


Figure 5
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  		Figure 5. Adenosine and IFN-{alpha} synergistically increase the IFN-{gamma} production of PBMC via the A3 receptor. This effect is independent from underlying infection with HCV; ongoing, antiviral treatment, IFN-{alpha}-based therapies; and other chronic inflammatory liver diseases such as PBC. PBMC were incubated for 24 h. IFN-{gamma} production was investigated by ELISPOT assays in all experiments. (A) Representative wells are shown. (B) SFUs of one representative experiments are shown. (C) Only stimulation of the A3 receptor increased the IFN-{gamma} production. (D) PBMC from different individuals were incubated for 24 h. IFN-{gamma} production was determined by ELISPOT assays. *, Spots were confluent in the ELISPOT assay representing >>50 spots. PEG, Pegylated.

PBMC were stimulated for 24 h with different doses of IFN-{alpha} and adenosine. Although adenosine in a concentration of 2 µM had no effect, 10 µM and 50 µM adenosine caused a highly significant stimulation of IFN-{gamma} production of IFN-{alpha}-stimulated PBMC. There was no inhibitory effect of adenosine (Fig. 5B) .

To investigate which adenosine receptor was responsible for this synergistic effect between IFN-{alpha} and adenosine, PBMC were incubated with IFN-{alpha} in the presence or absence of the three adenosine receptor agonists CCPA, CGS, and IB-Meca, as shown in Figure 5C . Neither the A1 receptor agonist CCPA nor the A2B receptor agonist CGS influenced IFN-{gamma} secretion, and the A3 receptor agonist IB-Meca increased the IFN-{gamma} production.

Thus, adenosine increased the IFN-{gamma} production of IFN-{alpha}-stimulated PBMC synergistically in a dose-dependent manner via the A3 receptor, but both substances also inhibited cell proliferation as shown before.

The synergistic effect of IFN-{alpha} and adenosine is independent from underlying HCV infection or ongoing IFN-{alpha} treatment
IFN-{alpha} is used frequently for the treatment of chronic viral hepatitis, and IFN-{gamma} production is considered to be of importance for treatment outcome [46 ]. Therefore, we asked whether the synergistic effect of adenosine and IFN-{alpha} is altered in patients with viral hepatitis, nonviral chronic inflammatory liver diseases, or ongoing antiviral treatment with PEG-IFN-{alpha} and ribavirin. As shown in Figure 5D , the highly significant increase in IFN-{gamma} spots was similar in healthy controls, untreated patients with HBV/HCV infection, patients with HCV infection receiving antiviral treatment with PEG-IFN-{alpha} and ribavirin, and patients with nonviral chronic inflammatory liver disease (PBC).

The increased IFN-{gamma} production of PBMC in response to adenosine and IFN-{alpha} is caused by NK cells
Intracellular cytokine stainings were performed to determine the PBMC cell subset responsible for the increased IFN-{gamma} production of adenosine and IFN-{alpha}-stimulated PBMC. Although the IFN-{gamma} production by CD4+ and CD8+ T cells was not altered in the presence of both agents, an approximate fourfold higher number of CD56+ cells stained positive for IFN-{gamma} in response to IFN-{alpha} and IB-Meca (Fig. 6 ).


Figure 6
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  		Figure 6. Adenosine A3 agonist and IFN-{alpha} synergistically increase the IFN-{gamma} production of the CD56+ cell fraction exclusively. PBMC were incubated in the presence of IB-Meca and IFN-{alpha} for 6 h at the indicated doses. IFN-{gamma} production was investigated by intracellular staining. Different cell populations were distinguished via surface staining for CD3, CD14, and CD56. Analysis via flow cytometry. FSC-H, Forward-scatter-height; FL1-H, fluorescence 1-height.

Activation of the A3 receptor in combination with IFN-{alpha} raises the CD107a expression of CD56+ cells but lowers the production of TNF-{alpha}
We also investigated whether other effector functions of NK cells are affected by adenosine and IFN-{alpha}. The expression of CD107a by CD56+ cells was enhanced after stimulating PBMC with IB-Meca and IFN-{alpha}, suggesting the cytotoxic activity of NK cells also enhanced by coincubation with both compounds (Fig. 7A ). In contrast, a reduction of intracellular TNF-{alpha} production was observed in cultures with adenosine and IFN-{alpha} as compared with stimulation with IFN-{alpha} alone (Fig. 7B) .


Figure 7
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  		Figure 7. Adenosine A3 agonist and IFN-{alpha} have different effects on the cytokine production and antigen expression of CD56+ cells. PBMC were incubated in the presence of IB-Meca and IFN-{alpha} for 6 h at the indicated doses. (A) CD107a expression or (B) TNF-{alpha} production was investigated by intracellular staining. The CD56 cell population was distinguished via surface staining. Analysis via flow cytometry. Iono, Ionomycin.


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DISCUSSION
 
Adenosine has been shown to act as an endogenous regulator of innate and adaptive immunity [1 ]. Several inhibitory effects of adenosine on different cells such as neutrophils [47 ], APC [48 ], mast cells [49 , 50 ], eosinophils [29 , 51 ], and lymphocytes [14 ] have been described. Here, we confirm the inhibitory effect of adenosine on PBMC by inducing apoptosis of activated human PBMC and by inhibiting proliferation of PBMC. On the other hand, we show that adenosine can also have stimulatory effects, as adenosine in combination with IFN-{alpha} synergistically increased the IFN-{gamma} production of NK cells. The inhibitory and the stimulatory mechanisms were induced mainly via the adenosine A3 receptor.

Previously, it has been shown that adenosine can prevent proliferation of anti-CD3-stimulated T cells and that killer T cells generated in the presence of adenosine display weaker cytotoxic activity [14 ]. Our findings are in line with these studies, also showing a reduced cytotoxic activity in the presence of adenosine and a dose-dependent reduction of IFN-{gamma} production of PHA-stimulated PBMC. Similar results were obtained by stimulating lymphocytes with other mitogens such as SEB or by using TCR-specific stimulation with MHC I-restricted peptides or proteins stimulating CD4 cells (F. Broelsch, F. Jeffe, H. Wedemeyer, unpublished observations). Importantly, the inhibition of the effector functions was achieved by rather low concentrations of adenosine, and adenosine concentrations of 5–50 µM are considered to be physiological in inflamed tissues [3 ] and within a tumor microenvironment [4 ].

The expression of adenosine receptors on activated T cells has been shown previously [52 ]. Gessi et al. [52 ] demonstrated a rapid increase, in particular, of A3 receptor expression of T cells during activation by PHA. Here, we provide functional evidence for the importance of A3 receptor expression on mitogen-stimulated PBMC, as the A3 receptor agonist IB-Meca but not A1 and A2 agonists inhibited PHA-activated PBMC. However, future studies will need to dissect in more detail the role of different adenosine receptors, as inhibitory effects in mice have been reported for NK cells and T cells to be mediated mainly via the A2 receptors [24 , 25 ]. A3 adenosine receptor ligation leads to inhibition of the adenylyl cyclase and activation of phospholipase C via interaction with Gi/o/q proteins [2 , 23 , 53 ]. However, immunosuppressive effects are likely to be mediated by an increase rather than a decrease of cAMP. It is possible that in A3, signaling differs between humans and mice and that immunosuppression is caused by another mechanism.

We did not only investigate the effect of adenosine on mitogen-stimulated PBMC but also were interested specifically in the effects of adenosines on IFN-{alpha}-stimulated PBMC. IFN-{alpha} is widely used for the treatment of different cancers and viral infections and is believed to activate effector functions of T cells and NK cells. In mice, type I IFN is a central mediator of NK cell antitumor responses [54 , 55 ]. Here, we describe that for human PBMC, IFN-{alpha} stimulated IFN-{gamma} production of NK cells almost exclusively. Importantly, adenosine did not inhibit the IFN-{alpha}-dependent production of IFN-{gamma} by NK cells but rather, increased IFN-{gamma} release further. This effect was again mediated via the A3 receptor.

What could be the physiological relevance of our findings in the setting of a viral infection? Type I IFNs are produced early during viral infections, leading to some level of resistance to viral replication in infected and uninfected cells by different mechanisms [56 ]. IFN-{alpha} activates NK cells, enabling killing of virus-infected cells. If adenosine and IFN-{alpha} are present in the inflamed tissue, our data suggest that the production of the antiviral cytokine IFN-{gamma} is increased further. Noncytolytic inhibition of viral replication by IFN-{gamma} has been shown for viruses such as HBV, HCV, HIV, Maedi Visna, and Borna disease virus [57 58 59 60 61 ]; thus, high concentrations of IFN-{gamma} locally, at the site of viral replication, should be of benefit for the host. On the other hand, if effector cells would proliferate too strongly and not undergo apoptosis, immune pathology would be the consequence. Type I IFNs and adenosine have antiproliferative properties and can induce apoptosis of activated T cells. Thus, our data suggest that adenosine contributes differentially to the regulation of immune responses during inflammatory processes by increasing effector functions of NK cells in combination with IFN-{alpha} but also by preventing overwhelming immune responses through inhibition proliferation and induction of apoptosis of activated lymphoid cells.

Importantly, constant exposure to high levels of IFN-{alpha} in vivo did not alter the synergistic in vitro effect of adenosine and IFN-{alpha}, as PBMC from patients with viral hepatitis undergoing antiviral therapy showed similar properties than PBMC from healthy controls. One may speculate that stimulation of the A3 receptor could potentially also be used therapeutically in individuals infected with viral hepatitis to increase the suboptimal response rates so far to IFN-{alpha} therapy. NK cells have been shown to be impaired in chronic HCV infection [62 63 64 65 ]. Targeting the A3 receptor during IFN-{alpha} therapy could overcome NK cell impairment in chronic HCV and thus, contribute to viral clearance.

Several limitations of our study and open issues need to be considered. First, we used much higher concentrations of IB-Meca than EC50 values described before [43 ]. However, it has to be considered that EC50 values were determined in transfected Chinese hamster ovary cells [66 ], which may have much higher levels of A3 receptor expression and not necessarily reflect effects in vivo. Here, we performed numerous dilution experiments and are convinced that the effects using an IB-Meca concentration of 20 µM are mediated mainly via the A3 receptor, as A1 and A2 receptor agonists showed almost no effect in our experiments. Moreover, we could show that the effect of IB-Meca is reduced by adding the A3 receptor antagonist MRS 1191. Second, we do not know to what extent the observed synergistic effect of IFN-{alpha} and adenosine applies only to IFN-{gamma} production of NK cells or also holds true for other effector functions. Although cytotoxicity, as determined by CD107a expression, was also increased, IFN-{alpha}-induced TNF-{alpha} production was reduced by adding IB-Meca. Thus, future studies will need to investigate the effects of IFN-{alpha} and adenosine on production of other cytokines and expression of surface effectors. Third, it needs to be considered that adenosine is also able to inhibit several steps during an inflammatory process via the A2A receptor, including costimulation, immune cell proliferation, cytotoxicity, antigen presentation, and cytokine production [10 , 25 , 67 ]. This has been shown in several transgenic mouse models, demonstrating an important role of the A2A receptor during inflammatory processes in the lung, gut, liver, heart, CNS, kidney, and joints [24 ]. Fourthly, the detailed mechanisms of how IFN-{alpha} is mediating the increased IFN-{gamma} production of NK cells are not known at present. We have already obtained some evidence that interaction with other cells types present in PBMC is of importance, as sorted NK cells do not show this up-regulation (K. A. Stegmann, H. Wedemeyer et al., manuscript in preparation). However, further studies are needed to explore the mechanisms in more detail.

In conclusion, here, we describe a novel effect of adenosine in combination with IFN-{alpha} on the function of NK cells. Future studies will have to investigate detailed mechanisms being responsible for the enhanced IFN-{gamma} production and explore possible therapeutic implications of our findings.


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ACKNOWLEDGEMENTS
 
The study was supported by BMBF grant 01KI0788 (H. W. and M. C.), by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 738, Project B2 (H. W.), and by the International Research Training Group 1273, funded by the German Research Foundation (Deutsche Forschungsgemeinschaft).


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received January 17, 2008; revised October 10, 2008; accepted October 23, 2008.


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