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The Journal of Immunology, 2005, 175: 4383-4391.
Copyright © 2005 by The American Association of Immunologists

Gs Protein-Coupled Adenosine Receptor Signaling and Lytic Function of Activated NK Cells1

Tatiana Raskovalova*, Xiaojun Huang*, Michail Sitkovsky{ddagger}, Lefteris C. Zacharia{dagger}, Edwin K. Jackson{dagger} and Elieser Gorelik2,*

* Department of Pathology and University of Pittsburgh Cancer Institute, {dagger} Department of Pharmacology and Medicine and Center for Clinical Pharmacology, University of Pittsburgh, Pittsburgh, PA 15213; and {ddagger} New England Inflammation and Tissue Damage Institute, Northeastern University, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1–/– and AdoRA3–/– mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adenosine is an endogenous purine nucleoside that is formed from ATP in both the extracellular and intracellular compartments, and intracellular adenosine is shunted into the extracellular space through membrane nucleoside transporters (1). Extracellular adenosine binds to adenosine receptors (AdoRs)3 that are expressed by diverse cell types and that mediate various biological effects of adenosine. Four different AdoRs have been identified (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3) that belong to the G protein-coupled seven transmembrane superfamily of cell surface receptors that are known as purinergic P1 receptors. AdoRA1 and AdoRA3 are negatively coupled to adenylate cyclase through the Gi/o protein {alpha}-subunits, whereas AdoRA2A and AdoRA2B are positively coupled to adenylate cyclase through Gs proteins. Therefore, AdoRA2A and AdoRA2B signaling elevates cAMP; in contrast, AdoRA1 and AdoRA3 activation inhibits cAMP production (2).

Extracellular adenosine mediates a variety of physiological effects in the nervous and cardiovascular systems (1). Adenosine constitutively present in the extracellular space of normal tissues at low concentrations. However, concentrations of adenosine quickly increase from nanomolar to micromolar concentrations in response to tissue hypoxia. Increased adenosine production under hypoxic conditions is due to inhibition of adenosine kinase and increased adenine nucleotide breakdown via the intracellular 5'-nucleotidase pathway. Adenosine markedly reduces damage induced by ischemia and neutrophil infiltration in many tissues (kidneys, lung, brain, spinal cord, and skin) (1, 3, 4, 5). Adenosine reduces hypoxia by increasing blood flow, and this response is mostly due to the vasodilatating properties of adenosine and by its ability to stimulate new blood vessel formation. In addition, adenosine inhibits inflammatory activity of neutrophils as well as macrophages and lymphocytes. Thus adenosine ameliorates inflammation in various diseases such as pleural inflammation, ischemia-reperfusion injury, rheumatoid arthritis, and endotoxin-mediated shock (1, 3, 4, 5). Adenosine also suppresses production of inflammatory cytokines such as TNF-{alpha} and IL-1 (5, 6, 7), and the anti-inflammatory effects of salicylates and methothrexate are largely mediated via stimulation of adenosine production (3, 8, 9). Thus by inhibiting the functional activity of lymphoid cells and the production of proinflammatory cytokines, adenosine protects ischemic tissue from excessive damage (3, 5, 6, 7).

Currently most studies are focused on the investigation of the anti-inflammatory properties of adenosine and its ability to protect normal tissues from hypoxia and inflammation. However, it is possible that adenosine plays an important role in protecting malignant tissue as well. Tumor cells are highly efficient in stimulating new blood vessel formation that is essential for tumor growth and metastatic dissemination. However, tumor growth still out paces blood vessel development, and therefore tumor growth is associated with tumor hypoxia as a result of insufficient vascularization, and this may trigger the production of adenosine by tumor cells. For example, analysis of the extracellular fluid of solid carcinomas shows that intratumor levels can reach as high 10 µM (10).

Increased intratumor levels of adenosine may have a protective effect by stimulating blood vessel formation and by inhibiting the function of tumor-infiltrating immune cells. Studies using allogenic specific CTLs from transgenic mice demonstrate that adenosine inhibits the cytotoxic activity of T lymphocytes. However, this inhibitory effect requires relatively high concentrations (50–5000 micromolar) of adenosine and 2-chloroadenosine (CADO, a metabolically more stable analog of adenosine) (11). It was demonstrated that CADO inhibits the major functions of CTLs such as TCR activation, cytotoxic granule release, TCR-triggered up-regulation of Fas ligand (FasL) and FasL-mediated lysis of target cells (11). Previous studies of the role of AdoRs in the regulation of the cytotoxic activity of T cells were based on the use of various AdoR agonists and antagonists, and results of such studies are controversial. Experiments with pharmacological antagonists of various AdoRs support the concept that the cAMP-elevating AdoRA2A is primarily responsible for inhibition of allospecific CTL cytotoxic activity (11, 12). However, no involvement of AdoRA2s in the inhibitory effects of CADO on the cytotoxic activity of anti-CD3-stimulated T cells was found (13). It was concluded that AdoRA3 is primarily responsible for the inhibitory effect of CADO on activation and generation of anti-CD3-activated T cells (13). However, CADO appears to inhibit the cytotoxic activity of anti-CD3-activated T cells via AdoRs that are not AdoRA1 or AdoRA2A (14).

These pharmacological studies have limitations due to the fact that agonists and antagonists are only selective, not specific, for their intended molecular target. Recently developed AdoR knockout (KO) mice are opening a new opportunity for the investigations of the biological significance of adenosine and its receptors in tumor biology. Currently mice with genetically disrupted AdoRA1, AdoRA2A, and AdoRA3 are available (15, 16, 17).

In the present study, we analyze the capacity of adenosine and its analogues to inhibit the ability of activated NK cells to kill tumor cells. To investigate the involvement of AdoRs in this suppressive effect of adenosine, IL-2-activated NK cells from AdoR KO mice were used. The rationale for using lymphokine-activated killer (LAK) cells was based on the following considerations: 1) although LAK cells are able to kill, both in vitro and in vivo, various human and murine tumor cells, the effects of adenosine on LAK cell cytotoxicity were unknown; 2) although in some cancer patients, LAK cells are able to reduce tumor load, they have limited therapeutic efficacy against large tumors in most terminally ill cancer patients (18, 19). It is therefore possible that the efficacy of LAK cells is inhibited by tumor-produced adenosine; 3) LAK cells can be easily generated from wild-type and AdoR KO mice, thus allowing for the analysis of the role of the individual AdoRs in the inhibitory effects of adenosine; and 4) previously we developed an approach that allows isolation and expansion of highly purified LAK cells based on the ability of IL-2 to increase the adhesion of activated NK cells to plastic. Thus by removal of nonadherent cells and culture of adherent cells in the presence of IL-2 a highly purified population of highly cytotoxic LAK cells can be isolated and expanded (20). We used this approach to analyze the ability of adenosine to inhibit the cytotoxic function of purified LAK cells.

Our studies demonstrate that adenosine and its analogues inhibit the ability of activated NK cells to destroy tumor cells. This inhibitory effect is rapid in onset and is induced at relatively low concentrations (0.7–5 µM) of CADO, and both perforin- and FasL-mediated cytotoxicity can be inhibited by adenosine. Studies with various agonists and antagonists of AdoRs indicate that the inhibitory effects of adenosine and CADO are mediated via cAMP-elevating AdoRA2As. Our studies also show that adenosine and adenosine analogues still suppress the cytotoxicity of LAK cells generated from AdoRA1 and AdoRA3 KO mice, whereas adenosine does not inhibit the cytotoxic activity of LAK cells from AdoRA2A KO mice. However, with very high concentrations of CADO, a relatively low level of inhibition of the effector function of LAK cells from AdoRA2A mice is observed. These data indicate that AdoRA2A signaling plays a predominant role in inhibition of LAK cell cytotoxicity, although low affinity AdoRA2B might have a contributing inhibitory effect at high concentrations of adenosine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Females (6- to 8-wk-old C57BL/6) were purchased from The Jackson Laboratory. AdoRA2A+/– mice were provided by Dr. Jiang Fan Chen (Northeastern University, Boston, MA) and were bred in our animal facility. AdoRA2A+/+ and AdoRA2A–/– mice were used in our experiments. The AdoRA2A genotypes of mice were determined by Southern blot analysis yielding the expected 7.5 and 5.0 kb restriction fragments for wild-type and mutant alleles, respectively (16). Experiments were performed in accordance with the approved institutional protocol and guidelines of the Institutional Animal Care and use Committee.

Reagents

Adenosine, CADO, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), CGS21680 (CGS), 5-N-ethylcarboxamide adenosine (NECA), MRS 1706, and forskolin were purchased from Sigma-Aldrich. NEMADO, ZM241385 (ZM) were obtained from Tocris.

Tumor cells

3LL Lewis lung carcinoma cell line was cultured in RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotics as described (21). These cells were used as the target for testing LAK cell-mediated cytotoxicity. To assess the effects of hypoxia on adenosine production by 3LL tumor cells, cells were cultured for 24 h in an air-tight chamber with inflow and outflow valves (Billups-Rothenberg). This chamber was infused with a gas mixture of 5% CO2 and 95% N2 (Valley National Gas) at a flow rate of 3 l/min for 15 min twice a day as described (22). This created hypoxia with ~1% oxygen. Medium conditioned by cells growing for 24 h was collected. Because of the very fast metabolism of adenosine by adenosine deaminase, EHNA, an inhibitor of adenosine deaminase, was added to the medium before harvesting at a final concentration of 30 µM. Condition medium collected from tumor cells cultured under hypoxic and normoxic conditions was frozen (–80°C) before testing the adenosine concentrations.

LAK cell generation

LAK cells were generated from spleens obtained from C57BL/6, AdoRA2A+/+, AdoRA2A–/–, AdoRA1+/+ and AdoRA1–/– and AdoRA3–/– mice. Spleens from AdoRA2A+/+ and AdoRA2A–/– were obtained from animals bred in our facility. Spleens from AdoRA1+/+ and AdoRA1–/– mice were obtained from animals bred by Dr. Patrick Kochanek (University of Pittsburgh, Pittsburgh, PA). The original breeder pairs were obtained by Dr. Kochanek from Dr. Jurgen Schnermann (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Spleens from AdoRA3–/– mice were obtained from AdoRA3–/– mice provided to Dr. Michael Sitkovsky by Dr. Marlene Jackobson (Merck). A single-cell suspension of spleen cells was prepared in complete RPM1 1640 medium supplemented with 10% FCS, nonessential amino acids, sodium private and 5 x 10–5 2-ME. RBC were lysed using red cell lysing buffer (Sigma-Aldrich). Spleen cells were cultured in T-75 flasks for 3 days in the presence of IL-2 (6000 IU/ml). Some experiments were performed using a bulk of IL-2-activated spleen cells. To obtaine purified fraction of LAK cells, spleen cells were cultured for 3 days with IL-2 and nonadherent spleen cells were removed and the flasks were washed with prewarmed (37°C) complete medium to remove cells that were not firmly attached to the plastic. Plastic adherent cells were cultured for an additional 3–8 days. This approach generates large numbers of purified highly cytotoxic LAK cells (20).

Cytotoxic activity of LAK cells

The cytotoxic activity of LAK cells was tested against 51Cr-labeled 3LL Lewis lung carcinoma cells. LAK cells were distributed into V-bottomed 96-well plates with and without test agents, and 10 min later, radiolabeled 3LL tumor cells (5 x 103/well) were added. LAK cell cytotoxicity was determined in triplicate at the various E:T ratios. After 4 h of incubation at 37°C, supernatants (25 µl) were transferred into yttrium silicate scintillator-coated white microplates (LumaPlate-96; PerkinElmer Life and Analytical Sciences), and the level of {beta}-emission by released 51Cr was measured in a beta counter. The percentage of cytotoxicity was calculated. The SD among the triplicates was always below 10% of mean. When multiple E:T ratios were tested, the cytotoxic activity was assessed by calculation of lytic units (LU) from regression curves plotted from the various E:T ratios (20). Data are expressed as LU 20% per 1 x 106 effector cells. One LU was defined as the number of effector cells required to lyse 20% of the target cells (20).

RT-PCR of AdoR gene expression in LAK cell

RT-PCR was performed using SuperScript one-step RT-PCR with platinum Taq. Total RNA was extracted from brain, thymus, and LAK cells generated from C57BL/6 mice and AdoRA2A–/– mice using TRIzol reagent according to the manufacturer’s specifications (Invitrogen Life Technologies). RT-PCR was conducted in an automatic DNA thermocycler (PerkinElmer Life and Analytical Sciences). All PCR primers of mouse AdoRs were synthesized by Sigma-Genosys Ltd according to the previously described sequences (13). cDNA synthesis and predenaturation was performed in 1 cycle at 50°C for 30 s and 94°C for 2 min that was immediately followed by PCR amplification. The amplification protocol for all AdoRs (40 cycles) was as follows: denaturation at 94°C for 15 s, annealing at 60°C for 1 min, extension at 72°C for 1 min, and final extension of 1 cycle at 72°C, 10 min. PCR products were visualized by electrophoresis across an ethidium bromide-stained 1.5% Tris-acetate-EDTA agarose gel, and the detected PCR amplicon was compared with a 100-bp ladder (Invitrogen Life Science). Steady-state expression of {beta}-actin was used to control for equal product loading.

Analytical method for adenosine and cAMP

Adenosine and cAMP concentrations in culture medium or tumor ascites were analyzed by HPLC using fluorescence detection as previously described by us (23). Briefly, ~40 µl of each sample were mixed with 5 µl of 9-D-arabinofuranosyladenine (internal standard; 10 µM) and 5 µl of 0.5 M acetate buffer (pH 4.8) and 4 µl of 50% chloroacetaldehyde in water. Samples were heated for 1 h at 80°C. Forty microliters of each sample were injected into an Isco high-pressure liquid chromatograph (pump model 2350, 4.6 x 250 mm C18 column), and the eluant was monitored with a Waters model 470 scanning fluorescent detector (wavelengths for excitation and emission were set at 275 nm and 420 nm, respectively). The flow rate was 1.2 ml/min, and the mobile phase was 95.5% citrate-phosphate buffer (0.014 M citric acid and 0.017 M Na2HPO4) and 4.5% acetonitrile.

cAMP production

LAK cells were distributed into 4-ml tubes (0.5 x 106 cells/0.5 ml/tube). LAK cells were incubated with ZM (0.3 µM) for 10 min and then CADO, CGS, or NECA was added. After 10 min of incubation, tubes were centrifuged, culture medium was removed, and 1 ml of ice-cold 1-propanol was added to cells. After shaking, cellular extracts were harvested, frozen, and later used for cAMP analysis using HPLC as described above.

Statistics

Statistical analysis of the data was performed using Student’s t test. The significance level was set up at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In vitro and in vivo adenosine production by tumor cells

We first evaluated the ability of hypoxia to stimulate adenosine production by 3LL Lewis lung carcinoma cells in vitro. 3LL tumor cells were distributed into 24-well plates (1 x 106cells/ml/well) and cultured for 24 h under normoxic (20% oxygen) and hypoxic (1% oxygen) environments. Medium conditioned by normoxic 3LL tumor cells contained 15 nM of adenosine per 1 x 106 cells per 24 h, whereas under hypoxic conditions adenosine concentrations in the medium increased nine times to 137.8 nM/1 x 106 cells/24 h.

To test the intratumor level of adenosine, BALB/c mice were inoculated i.p. with MethA sarcoma cells (1 x 106). One week later, the ascitic fluid was harvested. To prevent adenosine metabolism ex vivo, ascites was collected into a syringe containing EHNA (an inhibitor of adenosine deaminase). HPLC analysis revealed that ascites contain high levels of adenosine (1968 ± 207 nM) as well as cAMP (9464 ± 134 nM). These results indicate that the concentration of adenosine in MethA ascites can reach ~2 µM. It was of interest to test whether adenosine at this concentration is able to inhibit the cytotoxic activity of IL-2-activated NK cells.

Effect of CADO on the generation of LAK cells and their effector function

Spleen cells were cultured for 3 days in the presence of IL-2 (6000 IU/ml). To test the ability of CADO to affect IL-2 activation, some spleen cells were cultured in the presence of 2 µM of CADO. After 3 days, CADO-treated and nontreated spleen cells were washed and mixed with 51Cr-labeled 3LL tumor cells at E:T ratios (50:1–12.5:1) and their cytotoxic activity was tested in the 4 h cytotoxicity assay. The results presented in Fig. 1A show that CADO significantly (p < 0.05) inhibited IL-2-induced activation of the cytotoxic activity of spleen NK cells. We next tested whether CADO is able to inhibit the effector function of already activated NK cells. For this, spleen cells activated with IL-2 for 3 days in the absence of CADO were incubated with radiolabeled 3LL tumor cells in the presence of a different concentration of CADO (0.7–32 µM). CADO even at 0.7 µM was able to significantly (p < 0.05) inhibit the cytotoxic activity of activated NK cells (Fig. 1B). This inhibitory effect was concentration-dependent and further increased with increased concentrations of CADO. To test whether the inhibitory effect of CADO is mediated via activation of AdoRA2s, LAK cell were preincubated for 10 min with ZM, an antagonist of AdoRA2s, and then mixed with CADO (2 µM) and radiolabeled 3LL tumor cells. CADO significantly (p < 0.05) inhibited LAK cell cytotoxicity and antagonist of AdoRA2s blocked this inhibitory effect of CADO (Fig. 1C). ZM alone did not affect the cytotoxic activity of LAK cells. These data suggest that the inhibitory effect of CADO on LAK cell cytotoxicity is mediated via AdoRA2s.



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  		FIGURE 1. The effect of CADO on NK cell activation and the effector function of activated NK cells. A, Effect of CADO on the IL-2 activation of NK cells. Spleen cells of C57BL/6 mice were culture with IL-2 (6000 IU/ml) in the presence or absence of CADO (2 µM). After 3 days, the cytotoxic activity of spleen cells was tested against radiolabeled 3LL tumor cells at E:T ratios of 50:1–12.5:1. The cytotoxic activity of LAK cells was evaluated by calculation of LU at 20% cytotoxicity per 1 x 106 LAK cells (LU20/1 x 106). CADO significantly (p < 0.05) inhibited IL-2 activation of spleen cells. B, Effect of CADO on the cytotoxic activity of IL-2-activated NK cells. Spleen cells cultured with IL-2 for 3 days were mixed with CADO (0.7–32 µM) and 51Cr-labeled 3LL tumor cells at the E:T ratios of 50:1–12.5:1. The levels of cytotoxicity were determined after 4 h of incubation at 37°C. LAK cell cytotoxicity was significantly (p < 0.05) inhibited at all tested concentrations of CADO. C, Blockade of the inhibitor effect of CADO by the AdoRA2A antagonist ZM. The cytotoxic activity of IL-2-activated NK cells was tested in the presence of CADO (2 µM) or CADO (2 µM) plus the AdoRA2 antagonist ZM (0.3 µM) or ZM alone. ZM significantly (p < 0.05) blocked the inhibitory effect of CADO.

 
We next tested the ability of CADO and adenosine to inhibit the cytotoxic activity of purified highly cytotoxic LAK cells. Spleen cells of C57BL/6 mice were cultured for 3 days with IL-2 (6000 IU/ml). Plastic nonadherent cells were removed, and a small proportion of adherent cells was cultured in the presence of IL-2 for an additional 3–7 days. This results in the accumulation of a large quantity of purified highly cytotoxic LAK cells (20). Their cytotoxic activity was tested at E:T ratio 20:1 in the presence or absence of CADO (2–64 µM). At all tested concentrations, CADO substantially inhibited LAK cell cytotoxicity (Fig. 2A). At the tested concentrations, CADO was not toxic to LAK or 3LL tumor cells. The inhibitory effect of CADO was rapid and was fully developed after 2 h of incubation with a mixture of LAK and tumor cells.



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  		FIGURE 2. The inhibitory effect of CADO (A) or adenosine (B) on LAK cytotoxicity. Plastic adherent IL-2-activated NK cells were expanded by cultured with IL-2 for 3 additional days. The cytotoxic effect of purified LAK cells was tested against radiolabeled 3LL tumor cells (E:T ratio 20:1) in the presence of different concentrations of CADO (A) or adenosine (B). Due to quick metabolization of adenosine by adenosine deaminase the effect of adenosine on LAK cytotoxicity was tested in the presence of 30 µM of EHNA, an inhibitor of adenosine deaminase. A reduction of LAK cell cytotoxicity was significant (p < 0.05) at all tested concentrations of adenosine or CADO.

 
Adenosine, in contrast to CADO, is quickly metabolized by adenosine deaminase. To assess the ability of adenosine to inhibit the ability of LAK cells to kill 3LL tumor cells, the cytotoxic activity of LAK cells was tested in the presence of adenosine and EHNA (30 µM), an inhibitor of adenosine deaminase. As shown in Fig. 2B, adenosine at all tested concentrations (2–100 µM) significantly (p < 0.05) reduced the cytotoxic activity of LAK cells. Adenosine alone or EHNA alone did not inhibit LAK cytotoxicity.

Effect of AdoR agonists CGS and NECA on cAMP production and LAK cell cytotoxicity

To further analyze the involvement of AdoRs in adenosine-mediated inhibition of LAK cell cytotoxicity, the effect of AdoR agonists on LAK cytotoxicity was evaluated. Purified LAK cells were mixed with 51Cr-labeled 3LL cells at an E:T ratio of 20:1 in the presence or absence of the highly selective AdoRA2A agonist CGS. After 4 h of incubation at 37°C, the percentage of cytotoxicity was calculated. In parallel, the ability of CGS to induce cAMP production by LAK cells was tested. The cAMP production was tested 10 min after LAK cell incubation with CGS. The AdoRA2A agonist CGS showed dose-dependent inhibition of LAK cytotoxicity (Fig. 3A). Higher concentrations of CGS showed higher production of cAMP that coincided with a higher inhibitory effect of CGS on LAK cell cytotoxicity. Similar experiments were performed using NECA, a nonselective agonist of AdoRA2As and AdoRA2Bs (12). The results demonstrate that NECA at concentrations of 250 and 400 µM almost completely inhibited LAK cell cytotoxicty (Fig. 2B). The inhibitory effects of NECA paralleled the high cAMP production (Fig. 2B). At lower concentrations (60–125 µM), NECA induced lower levels of cAMP and manifested lower levels of inhibition of LAK cell cytotoxicity (Fig. 3B). No direct cytotoxic effect of CGS or NECA on LAK or tumor cells was observed. Thus, our results suggest that the inhibitory effect of adenosine is mediated via cAMP-elevating AdoRA2A signaling.



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  		FIGURE 3. Inhibitory effect of nonselective AdoRA2-selective agonist CGS (A) and nonselective AdoRA2A agonist NECA (B) on LAK cytotoxicity. LAK cells were mixed with radiolabeled 3LL tumor cells (E:T ratio 20:1) and different concentrations of NECA or CGS. After 4 h of incubation, the level of cytotoxicity was determined. To assess cAMP production, LAK cells were incubated with CGS and NECA; 10 min later, ice-cold 1-propanol was added, and cAMP levels in the cell extract were measured by HPLC. {triangleup}-{triangleup}, cAMP; {blacksquare}-{blacksquare}, percentage of cytotoxicity.

 
To further confirm the involvement of AdoRA2s in the inhibitory effect of adenosine and its analogues, it was of importance to demonstrate the ability of antagonists of AdoRA2 to block their inhibitory effects. However, in several our experiments AdoRA2 antagonist ZM failed to block the inhibitory effect of NECA on LAK cell cytotoxicity. Therefore, we compared the ability of ZM to block NECA-induced cAMP production and LAK cytotoxicity. LAK cells were incubated with NECA at different concentrations (7–400 µM). In parallel, LAK cells were preincubated with ZM (0.3 µM) and then with NECA. After 10 min, cAMP was extracted and analyzed by HPLC. NECA induced cAMP in a concentration-dependent manner (Table I). ZM pretreatment blocked NECA-induced cAMP production. This blocking effect was dependent on the concentration of NECA. At low concentrations of NECA (15–30 µM), ZM completely blocked NECA-induced cAMP production (Table I). However, at high concentrations of NECA (60–400 µM), ZM only partially reduced cAMP production in LAK cells. This residual cAMP might still be immunosuppressive and may prevent full restoration of LAK cell cytotoxicity by ZM. Indeed, ZM reduced cAMP from 48.4 to 37.3 pM in LAK cells treated with 400 µM of NECA, but it was not sufficient to abrogate the immunosuppressive effect of NECA (Table I). At 60 µM, NECA produced less cAMP and had a less profound inhibitory effect on LAK cell cytotoxicty. In the presence of ZM, cAMP production was reduced from 9.7 to 2.8 pM, and in parallel, ZM partially blocked the inhibitory effect of NECA and increased LAK cell cytotoxicity. The blocking effect of ZM was more prominent at 15 µM NECA where it reduced cAMP to nondetectable levels and almost completely restored LAK cell cytotoxicity (Table I).


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  		Table I. The effect of ZM on NECA-induced cAMP production and LAK cell cytotoxicity

 
The effect of CADO on perforin- and FasL-mediated cytotoxicity

LAK cells kill target cells via perforin/granzyme- and/or via FasL-mediated cytotoxicity (24, 25, 26). We tested which lytic mechanisms are affected by adenosine. In this regard, LAK cells were generated from FasL KO (gld) mice or from perforin KO mice. At E:T ratio 20:1 LAK cells generated from wild-type and FasL KO mice showed a high level of cytotoxicity. CADO at 2–50 µM substantially inhibited the cytotoxic activity of LAK cells of wild-type and FasL KO mice (Fig. 4A). Similarly, the nonselective AdoRA2 agonist NECA (50 µM) and CGS (30 µM), a selective agonist of AdoRA2A, inhibited the cytotoxic activity of LAK cells from FasL KO and wild-type mice (Fig. 4B). These experiments demonstrate that in the absence of FasL, LAK cells kill tumor cells by perforin/granzyme-mediated mechanism that can be inhibited by CADO and AdoRA2A agonists.



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  		FIGURE 4. Inhibition of perforin- and FasL-mediated cytotoxicity by CADO. A, The cytotoxic effect of LAK cells from control and FasL KO mice was tested against radiolabeled 3LL tumor cells (E:T ratio 20:1) in the presence of 2–50 µM of CADO. B, Effect of NECA (50 µM), CGS (30 µM), and CADO (2 µM) on the cytotoxic activity of LAK cells from FasL+/+ and FasL–/– mice (E:T ratio 20:1). C, Fas expression by 3LL tumor cells. 3LL tumor cells were stained with anti-CD95-PE mAb and analyzed by flow cytometry. Second peak represents the distribution of Fas-positive 3LL tumor cells. D, Effect of adenosine + EHNA and CADO on the cytotoxic activity of LAK cells generated from perforin–/– mice (E:T ratio 100:1). A reduction of LAK cell cytotoxicity was significant (p < 0.05) at all tested concentrations.

 
LAK cells generated from perforin KO mice showed low cytotoxicity against 3LL cells killing only 4.4% of 3LL tumor cells at the E:T ratio 20:1. Therefore, LAK cells from perforin KO mice were tested at higher E:T ratio (100:1) and they were able to kill 21.7% of 3LL tumor cells, which was probably generated via FasL triggering apoptosis of 3LL tumor cells. Our flow cytometric analysis of Fas expression by 3LL cells revealed that 76.4% of 3LL tumor cells express Fas (Fig. 4C). Lack of Fas expression by tumor cells does not preclude possibility of Fas-mediated lysis because incubation of tumor cells with LAK cells leads to up-regulation of Fas (26). However, this killing was usually observed in 18-h cytotoxicity assay. It was shown that LAK cells from perforin-deficient mice could lyse in a 4 h 51Cr-release assay only target cells that express high levels of Fas molecules (26). High level of Fas expression by 3LL tumor cells and the ability of LAK cells from perforin-deficient mice to kill 3LL tumor cells in a short 4-h cytotoxicity assay indicate that this lysis is Fas-mediated. We tested whether adenosine could inhibit Fas-mediated lysis. In the presence of adenosine (15 and 50 µM) and inhibitor of adenosine deaminase EHNA (30 µM), the cytotoxic activity of perforin-deficient LAK cells was significantly (p < 0.05) inhibited (Fig. 4D). Similarly, CADO inhibited the cytotoxic activity of these LAK cells (Fig. 4D). Thus, these data indicate that adenosine and its agonists are able to inhibit perforin- and FasL-mediated cytotoxic activity of LAK cells.

Effect of the genetic deletion of AdoRs on the inhibitory effects of CADO

Recently generated AdoR KO mice provide a unique opportunity to directly and precisely characterize the role of each individual AdoR in the regulation of the cytotoxic activity of killer cells. To clarify the role of cAMP elevating AdoRA2As in regulation of LAK cell cytotoxicity, we generated LAK cells from spleen cells of AdoRA2A+/+ and AdoRA2A –/– KO mice. We analyzed the profile of AdoR expression in these LAK cells using RT-PCR. RNA from brain cells was used as a positive control because these cells express all four AdoRs. Thymocytes express all AdoRs but showed a low level of A1 expression. LAK cells from wild-type mice expressed all receptors, except AdoRA1. LAK cells from AdoRA2A KO mice, as expected, showed no expression of A1 and A2A, and a reduced level of AdoRA2B message was found (data not shown).

We tested the ability of CADO to inhibit the cytotoxic activity of LAK cells generated from spleen cells of AdoRA2A–/– and AdoRA2A+/+ mice. LAK cells were mixed with 51Cr-labeled 3LL tumor cells at E:T ratios 6:1 in the presence or absence of CADO (2 µM). In some groups, a specific antagonist of AdoRA2Bs (MRS 1706 (0.3 µM)) or ZM (0.1 µM), a nonspecific antagonist of AdoRA2, was added 10 min before CADO. CADO significantly (p < 0.001) inhibited the cytotoxicity of LAK cells from AdoRA2A+/+ mice. MRS1706, an antagonist of AdoRA2Bs, only slightly attenuated CADO-induced inhibition, whereas ZM, an antagonist of AdoRA2As and AdoRA2Bs more efficiently blocked the inhibitory effect of CADO (Fig. 5A). The cytotoxicity of LAK cells from AdoRA2A–/– KO mice was not inhibited by CADO. Thus, experiments with LAK cells from AdoRA2A KO mice provided direct evidence that AdoRA2As play the predominant role in the inhibitory effect of adenosine, although the involvement of AdoRA2Bs cannot be excluded.



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  		FIGURE 5. Effect of CADO on the cytotoxic activity of LAK cells derived from AdoRA2A+/+ and AdoRA2A–/– mice. A, The cytotoxicity of LAK cells (E:T ratio 6:1) was tested in the presence of 2 µM of CADO and AdoRA2B-specific antagonists MRS 1706 (0.3 µM) or AdoRA2A nonselective antagonist ZM (0.1 µM). *, Significantly (p < 0.05) differs from other groups. B, The cytotoxicity of LAK cells from AdoR-A2A+/+ and AdoRA2A–/– mice was tested against 3LL cells in the presence of 5 or 15 µM of CADO and antagonist ZM (0.1 µM). The cytotoxic assay was performed at different E:T ratios (12:1, 6:1, and 3:1) and LU at 20% cytotoxicity per 1 x 106 LAK cells (LU20/106) were calculated. *, Significantly (p < 0.05) differs from other groups.

 
Because the AdoRA2B has a lower affinity for adenosine, the experiments were repeated with higher concentrations of CADO (5–15 µM). At 15 µM, CADO showed strong inhibition of the cytotoxic activity of LAK cells from AdoRA2A+/+ mice (Fig. 5B). The antagonist ZM (0.1 µM) only partially blocked the inhibitory effect of high concentrations (15 µM) of CADO but was able to block the inhibitory effect of 5 µM CADO (Fig. 5B). LAK cells from AdoRA2A–/– mice were completely resistant to the inhibitory effect of CADO at 5 µM. However, CADO at 15 µM was able to inhibit cytotoxicity, although this inhibition was only ~30%, whereas the cytotoxic activity of LAK cells from wild-type mice was reduced by 64% (Fig. 5B).

Our dose titration studies revealed that increased concentrations of CADO (16–64 µM) demonstrated increased levels of inhibition of the cytotoxic activity of LAK cells from AdoRA2A KO mice. However, these inhibitory effects were less profound than those found with LAK cells from wild-type mice. CADO at 64 µM inhibited the cytotoxic activity of AdoRA2A–/– LAK cells to the same degree as did CADO at 2 µM in AdoRA2A+/+ LAK cells (data not shown).

These results indicate that high affinity AdoRA2A plays a predominant role in the inhibitory effect of CADO on LAK cells. It is possible that the low affinity AdoRA2B could also provide an inhibitory signal and could be responsible for the inhibitory effect of high concentrations of CADO applied against LAK cells from AdoRA2A KO mice. Therefore, we next compared the inhibitory effect of NECA on LAK cells generated from control and AdoR2A KO mice. NECA is a nonselective agonist of AdoRA2s and stimulates both AdoRA2As and AdoRA2Bs. NECA markedly inhibited LAK cells from wild-type mice. However, NECA even at high concentrations (250–125 µM) only slightly inhibited the cytotoxic activity of LAK cells from AdoRA2A KO mice (Fig. 6A). This can be explained by the fact that in control mice NECA provides inhibitory signaling via both AdoRA2As and AdoRA2Bs, whereas in AdoRA2A–/– LAK cells only via AdoRA2Bs. Thus, these experiments demonstrate the prime importance of the AdoRA2A in the inhibitory effects of adenosine, although signaling via AdoRA2B makes a minor contribution.



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  		FIGURE 6. Effect of nonselective AdoRA2 agonist NECA (A) or adenylate cyclase activator forskolin (B) on the cytotoxic activity of AdoRA2A+/+ and AdoRA2A–/– LAK cells. The cytotoxic activity of LAK cells from AdoRA2A+/+ ({blacksquare}) and AdoRA2A–/– ({triangleup}) mice was tested against 51Cr-labeled 3LL cells at E:T ratio 12:1 in the absence and presence of NECA (250–7 µM) (A) or forskolin (25–0.3 µM) (B) in the 4-h cytotoxicity assay. *, Significantly (p < 0.05) differs from AdoRA2A+/+ group.

 
The inhibitory effect of adenosine is mediated via AdoRA2A signaling leading to adenylate cyclase activation and production of cAMP. It was of interest to test whether resistance to the inhibitory effect of adenosine in LAK cells from AdoRA2A KO mice is due to lack of signaling or impairment to downstream responses to cAMP. To test this, we examined the ability of forskolin to inhibit the cytotoxic activity of these LAK cells. Forskolin induces cAMP by directly activating adenylate cyclase independently of AdoRs. Forskolin (0.3–25 µM) inhibited LAK cell cytotoxicity in a concentration-dependent manner. The inhibitory effect of forskolin was similar in LAK cells from wild-type and AdoRA2A–/– mice (Fig. 6B). These results indicate that lack of AdoRA2A signaling is of prime importance in resistance of AdoRA2A-deficient LAK cells to the inhibitory effects of CADO.

To analyze possible involvement of other AdoRs in adenosine-mediated inhibition of LAK cytotoxicity, we generated LAK cells from AdoRA1 KO and AdoRA3 KO mice. CADO similarly inhibited the cytotoxic activity of wild-type, AdoRA1–/–, and AdoRA3–/– LAK cells (Fig. 7A). When LAK cells from wild-type, AdoRA1 KO, and AdoRA3 KO mice were treated with NECA also similar inhibitory effects were observed (Fig. 7B). These results indicate that adenosine-mediated inhibition of LAK cell activity is independent of AdoRA1 and AdoRA3 but mostly mediated via AdoRA2A signaling.



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  		FIGURE 7. Inhibitory effects of CADO (A) or NECA (B) on the cytotoxic activity of LAK cells generated from AdoRA1 and AdoRA3 KO mice. The cytotoxic activity of LAK cells from AdoRA1–/– ({triangleup}), AdoRA3–/– (X), and control wild-type+/+ ({blacksquare}) mice was tested against 51Cr-labeled 3LL cells at E:T ratio 12:1 in the absence and presence of CADO (2–64 µM) (A) or NECA (250–7 µM) (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our data indicate that adenosine and CADO substantially inhibit the cytotoxic activity of IL-activated NK cells against 3LL Lewis lung carcinoma cells. Similarly, CADO and adenosine were able to inhibit LAK cell cytotoxic activity against other targets, such as YAC-1 and B16 melanoma (data not shown). Although previous studies demonstrate the ability of adenosine and CADO to inhibit the cytotoxic activity of murine allogenic specific CTLs or CD3-activated T cells, relatively high concentrations (50–5000 µM) (11, 13, 14) are required. Importantly, the present study shows that much lower concentrations of CADO (0.7–5 µM) significantly inhibit the ability of the LAK cell to kill tumor cells. Moreover, our studies demonstrate that the concentration of adenosine in MethA ascites reaches 2 µM, and other studies indicate that the concentration of adenosine in solid tumors achieves levels as high as 13 µM (10). Therefore, it is likely that intratumor adenosine could inhibit the cytotoxic activity of tumor-infiltrated NK cells.

Analysis of the mechanisms of the inhibitory effects of CADO revealed that in CTLs, CADO inhibits cytolytic granule exocytosis as well as FasL expression (11). In agreement with these results in CTLs (11), we find that adenosine, CADO, and the AdoRA2 agonists CGS and NECA inhibit both perforin- and FasL-mediated mechanisms of LAK cytotoxicity.

Our experiments with AdoRA2 agonists CGS and NECA demonstrate that signaling via AdoR2s is primarily responsible for inhibition of the cytotoxic activity of LAK cells. The level of inhibition parallels the ability of these agonists to induce cAMP. The importance of cAMP in the inhibition of LAK cell cytotoxicity is further supported by the observation that direct activation of adenylate cyclase and production of cAMP by forskolin also results in inhibition of LAK cell cytotoxicity.

The involvement of cAMP-elevating AdoRA2s in the inhibitory effect of adenosine is further supported by our findings that ZM, a nonselective antagonist of AdoRA2s, blocks the inhibitory effects of CADO and NECA. Our analysis of the cAMP production by LAK cells treated with NECA demonstrates that high concentrations of NECA (400–125 nM) induce very high levels of cAMP. In this setting, although ZM reduces NECA-induced cAMP, residual concentrations of cAMP remain relatively high and could still inhibit LAK cell cytotoxicty. As a result, ZM does not completely block the inhibitory effects of high concentrations of NECA on LAK cell cytotoxic activity. With lower concentrations of NECA, cAMP production is lower and is further reduced in the presence of ZM. Under this condition, the inhibitory effects of NECA on LAK cytotoxic activity were completely blocked.

Our experiments with AdoRA2A KO mice brought more direct confirmation of the predominant role of AdoRA2As in adenosine-mediated inhibition of cytotoxic activity of LAK cells. However, with high concentrations (15–64 µM) some inhibitory effects of CADO are observed in AdoRA2A KO LAK cells, although the level of inhibition of cytotoxicity of LAK cells from these mice is much lower than that from wild-type mice. These findings suggest some involvement of low-affinity AdoRA2Bs that contribute to the inhibitory effects of CADO only at a higher concentration of CADO. More direct evidence of the importance of AdoR2Bs in the immunosuppressive effects of adenosine must await the availability of AdoRA2B KO mice.

Analysis of the effects of CADO on LAK cells from mice with genetically disrupted AdoRA1s and AdoRA3s reveals that lack of these receptors does not affect the ability of CADO to inhibit LAK cell cytotoxicity. Moreover, our RT-PCR analysis of AdoR expression in LAK cells shows that these cells do not express AdoR1s and that the expression of the AdoRA3 was lower, compared with the expression of AdoRA2. Therefore, AdoRA1s and AdoRA3s have no effect on the ability of CADO to inhibit LAK cell activity.

In summary, our experiments demonstrate that adenosine could be an important factor blocking the ability of LAK cells to kill tumor cells. The cAMP-elevating AdoRA2A plays a predominant role in this inhibitory effect of adenosine. In contrast, there is no evidence of the involvement of cAMP inhibitory AdoRA1s or AdoRA3s in the regulation of the cytotoxic activity of LAK cells. Adenosine should be considered an important intratumor factor that inhibits the effector function of NK and T cells and protects tumors from immune destruction. Although several intratumor factors with immunosuppressive activity have been identified, such as IL-10, TGF-{beta} these factors mostly inhibit generation of immune cells. In this regard, adenosine is unique and is able to inhibit the cytolytic function of already generated antitumor immune cells. Because the inhibitory effect of adenosine is mediated via AdoRA2s, antagonists of AdoRA2 could be used to increase the efficacy of antitumor immunotherapy.


   Acknowledgments
 
We thank Drs. Jiang Fan Chen (Northeastern University, Boston), Patrick Kochanek (University of Pittsburgh), and Marlene Jackobson (Merck, White House Station, NJ) for providing AdoR KO mice.


   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 These studies were supported by grants from the U.S. Army Breast and Prostate Cancer Research Program (DAMD17-03-1-0599 and DAMD-02-1-0127) (to E.G.) and National Institutes of Health Grant DK68575 (to E.K.J.). Back

2 Address correspondence and reprint requests to Dr. Elieser Gorelik, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Room 1.46, 5117 Centre Avenue, Pittsburgh, PA 15213. E-mail address: gorelik{at}pitt.edu Back

3 Abbreviations used in this paper: AdoR, adenosine receptor; CADO, 2-chloroadenosine; EHNA, erythro-9-(2-hydroxy-3-nonyl) adenine; LAK, lymphokine-activated killer; KO, knockout; FasL, Fas ligand; CGS, CGS21680; NECA, 5-N-ethylcarboxamide adenosine; ZM, ZM241385; LU, lytic units. Back

Received for publication May 19, 2005. Accepted for publication July 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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