Abstract
Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5′-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue’s metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.
Cellular and humoral immunity and their effector cells are regulated by APCs, the Th cell subtypes Th1 and Th2, and by their secreted soluble messengers (1, 2 ). Th1 cells primarily secrete IFN-γ, IL-2, and TNF-β, which promote cellular immunity, whereas Th2 cells secrete a different set of cytokines, primarily IL-4, IL-10, and IL-13, which promote humoral immunity (1, 2).
IL-12 is a 75-kDa heterodimeric cytokine composed of p35 and p40 chains; p35 is constitutively expressed in most cells, whereas p40 is up-regulated in monocytes and macrophages in response to infection or appropriate activating substances such as LPS (3). IL-12 is a central inducer of Th1 responses and cell-mediated immunity. First, it induces IFN-γ production from NK and T cells, which contributes to phagocytic cell activation and inflammation. Second, it favors Th1 cell differentiation and proliferation (3). IL-12 is essential for the clearance of certain intracellular pathogens such as Mycobacterium tuberculosis (4). However, excessive production of IL-12 may be responsible for the proinflammatory activities and the tissue damage typical of organ-specific autoimmunity, as seen in insulin-dependent diabetes mellitus in nonobese diabetic mice (5) and in type II collagen-induced arthritis in DBA/1 mice (6). In addition, stimulation of IL-12 secretion by microbial products was shown to be the crucial factor for the proliferation and differentiation of pathogenic autoreactive Th1 effector cells in experimental allergic encephalomyelitis (7).
Adenosine (ADO),2 together with ADP and ATP, belongs to a class of endogenous purine nucleosides produced by many cells during normal metabolic activity (8, 9, 10). These ubiquitous molecules are utilized in selective extracellular signaling (8, 9, 10). For example, ADO appears to be an important endogenous regulator of coronary blood flow (8). Postganglionic sympathetic nerve terminals also release ATP that is rapidly degraded to ADO, which induces vasodilatation mediated by A2 receptors (10). Local extracellular ADO levels increase dramatically during ischemia and hypoxia, possibly providing cytoprotection and increased blood flow to ischemic tissues (11). Inflammation and tissue injury represent pathologic states that are also associated with enhanced extracelluar ADO concentrations.
One of the first clinical observations linking ADO to the immune system was made in patients with SCID. These patients lack the enzyme adenosine deaminase, i.e., they are unable to catabolize ADO (12). This abnormality results in highly elevated plasma levels of ADO and consequent impairment of cellular immunity, marked by recurrent infections. The immunosuppression observed in SCID is explained by the direct lymphotoxicity of ADO catabolites and by the strong extracellular ADO-induced inhibition of the TCR-triggered proliferation and T cell effector functions mediated by A2a receptor signaling (13, 14). More recently, it has been demonstrated that ADO also exerts diverse anti-inflammatory effects, mediated mainly by A2 receptors, including diminished leukocyte accumulation, inhibition of C2 production, and reduction of the superoxide anion generation (15, 16, 17). Therefore, the regulation of ADO receptors is recognized as having significant therapeutic potential (9).
Recent evidence suggests that the anti-inflammatory effects of methotrexate and sulfasalzine, currently the most commonly prescribed and effective second-line agents for the treatment of rheumatoid arthritis (RA), are mediated in part by increases in the extracellular concentrations of ADO (15, 18). Because excessive production of IL-12 appears to play an important role in RA, the question arises: does ADO suppress IL-12 production? This may explain, at least in part, the beneficial effects of these drugs in RA. This hypothesis arose from previous studies showing that ADO inhibits the production of TNF-α (19, 20, 21, 22), another proinflammatory cytokine implicated in the pathogenesis of RA, while it increases the secretion of the anti-inflammatory cytokine IL-10 (23, 24), which suppresses IL-12 and TNF-α release. In this paper, we demonstrate that ADO analogues, via activation of A2a receptors on monocytes, cause substantial inhibition of human IL-12 production and simultaneous stimulation of IL-10 production. Through these mechanisms, increased concentrations of ADO may polarize the Th1/Th2 balance toward Th2 dominance and may contribute to the immunomodulatory properties of ADO.
Materials and Methods
Drugs and reagents
LPS from Escherichia coli, serotype K-235 (Sigma, St. Louis, MO), was dissolved in distilled water. The mixture was sonicated for at least 3 min in a sonicating bath, and aliquots were stored at −20°C until they were used. After thawing, appropriate dilutions were made in RPMI 1640 medium. Stapholococcus aureus Cowan strain 1 (SAC) was purchased from Calbiochem (La Jolla, CA). 5′-N-ethylcarboxamidoadenosine (NECA), N6-Benzyl-5′-N-ethylcarboxamidoadenosine (N6-Benzyl-NECA), 2-[p-(2-carbonyl-ethyl)-phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine (CGS-21680) HCL, 2-Chloro-N6-cyclopentyladenosine (CCPA), N6-(3-iodobenzyl)-adenosine-5′-N
Blood donors
Twenty healthy female and male volunteers participated in this study, which was approved by an institutional review board of the National Institutes of Health. Volunteers refrained from using any drugs, including cyclooxygenase inhibitors, or hormones for 1 wk before the study.
Whole blood cultures
Blood was drawn into sodium heparin-containing sterile blood collecting tubes (Vacutainer, Becton Dickinson, Lincoln Park, NJ) and diluted 1:5 with RPMI 1640 (supplemented with 1% glutamine and 50 μg/ml gentamicin) with no added exogenous serum. The blood (1 ml) was aliquoted in 24-well culture plates (Costar, Cambridge, MA). Bacterial LPS was added to allow a final concentration of 1 μg/ml for the induction of cytokines, and the samples were incubated in 5% CO2 at 37°C for 18 h. ADO analogues and receptor agonists were added to the wells 10 min before LPS or SAC stimulation. CSC, Rp-cAMPS triethylamine, and the phosphodiesterase inhibitor Ro 20-1724 were added 10 min before the ADO receptor agonists. After incubation, the blood was centrifuged, and the supernatant plasma was collected and stored in polypropylene tubes at −70°C until the samples were measured.
Monocyte elutriation and culture
Human monocytes were obtained from healthy volunteers of the National Institutes of Health, Department of Transfusion Medicine, Cell Processing Section. Mononuclear cells were isolated on lymphocyte separation medium and the monocytes were purified by counterflow centrifugal elutriation as previously described (25). The monocytes were cultured at 5 × 105 cells/ml. All cultures were set up in RPMI 1640 medium supplemented with 15% FCS, 1% glutamine, and 50 μg/ml gentamicin to yield a final volume of 1.5 ml. Cytokine production was induced by LPS (1 μg/ml), and all cultures were incubated in 5% CO2 at 37°C for 18 h. In several experiments, anti-IL-10 Abs were added at a concentration of 10 μg/ml. According to the manufacturer’s instruction, this concentration gives a 50% neutralizing dose in the presence of 5 ng/ml of rhIL-10.
Cytokine assays
Detection of IL-12 p35 and p40 mRNA
Human monocytes were cultured at 1.5 × 107 cells, with or without LPS (1 μg/ml), in the presence or absence of 10−5 or 10−6 M concentrations of the A2a agonist CGS-21680 at 37°C for 18 h. Total RNA was isolated by using TRIZOL reagent according to the manufacturer’s instructions. cDNAs were reverse transcribed with TaqMan reverse transcription reagents (Applied Biosystems). cDNA solution (5 μl) was used to quantitate mRNA levels using TaqMan cytokine gene expression plate I (Applied Biosystems)with 65 PCR cycles on the ABI PRISM 7700 sequence detection system (Applied Biosystems). Results were analyzed with the sequence detection system software.
Statistical analysis
All data are presented as mean ± SE. Statistical analysis was performed by one-way ANOVA.
Results
The ADO analogue, NECA, suppresses LPS-induced IL-12 and stimulates IL-10 production
Because ADO has an extremely short half-life and is rapidly metabolized, in our experiments we used nonhydrolyzable ADO analogues or more metabolically stable analogues that are able to mimic the effects of extracellular ADO (8, 9). The addition of increasing concentrations of the ADO analogue NECA to the LPS-stimulated whole blood cultures resulted in a dose-dependent inhibition of IL-12 p70 production (Fig. 1⇓A). NECA at a concentration of 10−5 M suppressed IL-12 production by 95%. However, increasing doses of NECA resulted in a dose-dependent increase of LPS-induced IL-10 production (Fig. 1⇓B), showing a substantial increase at a concentration of 10−6 M (a 240% increase).
Effect of the ADO analogue NECA on LPS-induced IL-12 p70 (A) and IL-10 (B) production in human whole blood from six normal volunteers. Increasing concentrations of NECA were added as indicated. Data are expressed as the mean ± SE. Mean LPS-induced IL-12 p70 and IL-10 concentrations were 54.2 ± 10.1 and 158.5 ± 51.4 pg/ml, respectively. Asterisks indicate significant differences at p < 0.05, compared with LPS control.
The effect of NECA on the IL-12 and IL-10 production is not stimulus-specific
To determine whether the effects of NECA on IL-12 and IL-10 production were limited to LPS stimulation, whole blood cultures were stimulated with SAC in the presence of increasing doses of NECA. As shown in Fig. 2⇓A, NECA strongly inhibited SAC-induced production of IL-12 p70 in a dose-dependent manner, while it simultaneously enhanced the production of IL-10 (Fig. 2⇓B).
Effect of the ADO analogue NECA on SAC-induced IL-12 p70 (A) and IL-10 (B) production in human whole blood from four normal volunteers. Increasing concentrations of NECA were added as indicated. Data are expressed as the mean ± SE. Mean LPS-induced IL-12 p70 and IL-10 concentrations were 22.7 ± 6.6 and 98.9 ± 11.26 pg/ml, respectively. Asterisks represent significant differences at p < 0.05, compared with LPS control.
ADO receptor agonists inhibit IL-12 and stimulate IL-10 production
The ADO analogue NECA is a nonselective agonist of A1, A2, and A3 receptors. To investigate whether the modulation of cytokine production is related to the stimulation of specific ADO receptors, the order of effectiveness of specific ADO receptor agonists was compared in LPS-stimulated whole blood. As evident from Fig. 3⇓A, the A2a receptor agonist CGS-21680 also exhibited strong dose-dependent inhibitory activity, whereas the A1 receptor agonist CCPA and the A3 receptor agonist N6-Benzyl-NECA were effective only at high concentrations. The median effective concentration for the A2a receptor agonist CGS-21680 (5 × 10−8 M) was approximately two orders of magnitude less than that for the A1 and A3 receptor agonists (10−6 and 5 × 10−6 M, respectively). The same order of effectiveness of ADO agonists was observed in their enhancing effect of IL-10 production (Fig. 3⇓B). These data suggest that the modulation of LPS-induced IL-12 and IL-10 production by ADO analogues in human whole blood is primarily mediated through A2a receptors.
Effect of the ADO receptor agonists CCPA (A1), CGS-21680 (A2a), and N6-Benzyl-NECA (A3) on LPS-induced IL-12 p70 (A) and IL-10 (B) production in human whole blood from seven normal volunteers. Increasing concentrations of ADO receptor agonists were added as indicated. Data are expressed as the mean ± SE. Mean LPS-induced IL-12 p70 and IL-10 concentrations were 74 ± 15.6 and 298 ± 78.2 pg/ml, respectively. Symbols represent significant differences at p < 0.05, compared with LPS control (♦, A1 agonist; ∗, A2a agonist; and +, A3 agonist).
Monocytes are the main targets for the effect of ADO analogues on IL-12 and IL-10 production in human peripheral blood
Monocytes are the major cellular source of IL-12 and IL-10 production in human whole blood (26, 27). To verify that peripheral blood monocytes were directly affected by ADO analogues, we stimulated enriched human monocytes with LPS in the presence of different concentrations of A1, A2, and A3 agonists. Although ADO receptor agonists had an inhibitory effect on the IL-12 p40 production, they potentiated IL-10 production in monocytes (Fig. 4⇓, A and B). Similar to the findings in whole blood, the A2a receptor agonist CGS-21680 expressed the strongest effects. The potency of two different A3 receptor agonists was also compared. Although IB-MECA induced a slightly stronger effect than N6-Benzyl-NECA, the effect of both A3 agonists was still much weaker than the effect of A2a receptor agonist CGS-21680. Because freshly isolated, unprimed human monocytes produce little IL-12 p70 when stimulated with LPS, the effect of ADO analogues on IL-12 p70 could not be reliably evaluated in these experiments.
Effect of the ADO receptor agonists CCPA (A1), CGS-21680 (A2a), N6-Benzyl-NECA (A3I), and IB-MECA (A3II) on LPS-induced IL-12 p40 (A) and IL-10 (B) production in human monocytes from six normal volunteers. Increasing concentrations of ADO receptor agonists were added as indicated. Data are expressed as the mean ± SE. Mean concentrations of IL-12 p40 and IL-10 were 1499.4 ± 320.3 and 715.7 ± 206.2 pg/ml, respectively. Symbols represent significant differences at p < 0.05, compared with LPS control (♦, A1 agonist; ∗, A2a agonist; +, A3I agonist; and ✤, A3II agonist).
CSC, an A2a-selective antagonist, blocks the effect of A1, A2, and A3 receptor agonists on the production of IL-12
As shown in Fig. 5⇓, the inhibitory effect of the A2a agonist CGS-21680 was blocked in the presence of the specific A2a antagonist CSC. Because the inhibitory effects of the A1 and A3 receptor agonists were observed only at high concentrations, we subsequently investigated the possibility that these effects may have resulted from nonspecific action via A2a receptors. As is visible in Fig. 5⇓, the A2a antagonist CSC was able to prevent the inhibitory effect of the A1 and A3 receptor agonists.
CSC, a selective A2a receptor antagonist (Ant), prevents the effect of A1 (CCPA), A2a (CGS-21680), and A3 (IB-MECA) receptor agonists (Ago) on LPS-induced IL-12 p40 production in human monocytes from six normal volunteers. CSC was added at a concentration of 10−6 M; A1, A2, and A3 Ago were added at a concentration of 10−6, 10−7, and 5 × 10−7 M, respectively. Data are expressed as the mean ± SE. Mean LPS-induced IL-12 p40 was 1792.7 ± 555.04 pg/ml. Asterisks represent statistically significant differences between the effect of A1, A2, and A3 Ago alone and their effect in the presence of the A2a receptor antagonist CSC. ∗, p < 0.06; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.
The A2a agonist CGS-21680 inhibits the expression of IL-12 p40 mRNA, induced by LPS
We examined the levels of IL-12 p35 and p40 mRNA in human peripheral monocytes with TaqMan cytokine gene expression plate I. As shown in Fig. 6⇓, A and B, LPS increased the levels of IL-12 p40 mRNA by 10-fold. Addition of the A2a agonist CGS-21680 dose-dependently suppressed LPS effect, suggesting that the effect of this drug is at the transcriptional level. On the other hand, the levels of IL-12 p35 mRNA were not affected by LPS and the A2a agonist.
Effect of the A2a agonist CGS-21680 on the levels of IL-12 p35 (A) and p40 (B) mRNA in human peripheral monocytes, induced by LPS. The levels of mRNA levels are shown as an n-fold increase compared with the levels of baseline condition, LPS(−). Data are expressed as the mean ± SE from three experiments with six measurements. Asterisk indicates a statistically significant effect of the A2a agonist CGS-21680 compared with LPS(+) (p < 0.01).
The A2a receptor agonist CGS-21680 mediates its effect through cAMP/protein kinase A (PKA) pathway
Stimulation of A2 receptors results in increased cAMP production (8, 9). To analyze the role of cAMP, we antagonized the effect of the A2a agonist CGS-21680 by application of Rp-cAMPS, a diastereomer of adenosine-3′,5′ cyclic monophosphothiate. Rp-cAMP is known to competitively inhibit the cAMP-induced activation of PKA. Monocytes were stimulated by LPS in the presence of A2a agonist CGS-21680 and, as shown in Fig. 7⇓A, CGS-21680 inhibited IL-12 production by 50%. The presence of Rp-cAMP together with the A2a agonist CGS-21680 reversed this inhibition dose-dependently. Phosphodiesterase is an enzyme involved in the breakdown of cAMP. Thus, inhibition of this enzyme results in an increase of cAMP levels. As shown in Fig. 7⇓B, the phosphodiesterase type IV inhibitor Ro 20-1724 had a dose-dependent inhibitory effect on the LPS-induced IL-12 production in the absence of CGS-21680. Moreover, the effect of Ro 20-1724 was additive to the effect of the A2a receptor agonist CGS-21680. Thus, the presence of increasing concentrations of Ro 20-1724 resulted in about a 2-fold potentiation of the inhibition of IL-12 production induced by CGS-21680. To further evaluate the role of cAMP in this mechanism, we added 8-(chlorophenylthio)-cAMP, a cAMP analogue, to our cultures. This drug dose-dependently inhibited LPS-induced IL-12 production, mimicking the effect of the A2a agonist CGS-21680 (Fig. 7⇓C).
Rp-cAMP triethylamine (Rp-cAMP, 100 and 300 μM) dose-dependently prevents the inhibition of CGS-21680 A2a Ago (10−7 M) on IL-12 p40 production in human monocytes from six normal volunteers (A). Dose-dependent effect of Ro 20−1724 (10−7, 10−6, 5 × 10−6, and 10−5 M) and its potentiation of the effect of CGS-21680 (10−7 M), a selective A2a receptor agonist (Ago), on IL-12 p40 production in human monocytes from six normal volunteers (B). The cAMP analogue 8-(chlorophenylthio)-cAMP dose-dependently inhibits LPS-induced IL-12 p70 production in whole blood from five normal volunteers (C). Data are expressed as the mean ± SE. Mean LPS-induced IL-12 p70 (Fig. 6⇑C) and IL-12 p40 (Fig. 6⇑, A and B) concentrations were 57.6 ± 17.1, 1083.2 ± 339.1, and 3190.6 ± 577.9 pg/ml, respectively. Asterisks in A indicate a statistically significant difference (p < 0.05). Asterisks in B indicate statistically significant differences of Ro 20-1724/CGS-21680 compared with Ro 20-1724 alone (p < 0.05). Pluses indicate significant differences of Ro 20-1724/CGS-21680 compared with CGS-21680 alone (p < 0.001). Asterisks in C indicate statistically significant difference (p < 0.01).
The effect of A2a receptor agonist CGS-21680 on IL-12 production is independent of endogenous IL-10 secretion
Previous studies indicate that IL-10 inhibits the secretion of proinflammatory cytokines from monocytes (28). These observations prompted us to consider the possibility that the inhibitory effects of A2 receptor agonists on IL-12 release were caused by induction of endogenous IL-10 production. Consequently, we investigated the effect of adding neutralizing IL-10 Abs to monocyte cultures stimulated with LPS in the presence of the A2a agonist CGS-21680. The mean LPS-induced IL-10 levels in our experiments were 871.2 ± 194.3 pg/ml. Thus, the concentration of endogenously produced IL-10 was ∼6-fold less than the concentration at which anti-IL-10 Abs express a 50% neutralizing dose (see Materials and Methods). We concluded that the concentration of neutralizing anti-IL-10 Abs used in these experiments was sufficient to eliminate endogenously produced IL-10. In the presence of 10−6 M CGS-21680, the LPS-induced IL-10 levels were 47.1 ± 10.1 pg/ml, whereas in the presence of anti-IL-10 Abs, the addition of this drug to the cultures resulted in IL-10 production of 41.6 ± 7.0 pg/ml (data not shown). These data suggest that endogenous production of IL-10 induced by LPS does not mediate the inhibitory effect of the A2a receptor agonist on IL-12 production.
Discussion
ADO is an endogenous nucleoside with immunoregulatory properties. The present study suggests that the inhibition of IL-12 production by ADO is an additional mechanism that may contribute to its anti-inflammatory and immunosuppressive effects. In this paper we demonstrate that the ADO analogues NECA and CGS-21680 mediate a strong and dose-dependent inhibition of LPS- and SAC-induced IL-12 production. The order of potency of NECA, CGS-21680, and A1 and A3 receptor agonists is: NECA = CGS-21680 ≫ CCPA > N6-Benzyl-NECA. Because A2a receptors are characterized by their high-affinity binding of the agonist CGS-21680, whereas A2b receptors display low affinity for NECA (9), this pharmacological profile of the response implies involvement of A2a receptors. This is further substantiated by the observation that the selective A2a antagonist CSC blocked the inhibitory effect of CGS-21680. Moreover, the inhibitory effect of CGS-21680 was potentiated by a phosphodiesterase inhibitor and prevented by an inhibitor of the type I and II PKA. Monocytes are the main source of IL-12 production in human whole blood (26, 27), and stimulation of A2 receptors in many cells, including monocytes, has been associated with increased generation of cAMP production (9, 16). Thus, ligand activation of A2a receptors through stimulation of the cAMP/PKA pathway appears to mediate the inhibition of IL-12 production by human monocytes. This is consistent with previous studies showing that ADO analogues inhibited, also via stimulation of A2 receptors, the secretion of the proinflammatory cytokine TNF-α by human monocytes (19, 20, 22).
In our experiments, the A1 and A3 receptor agonists had an inhibitory effect on IL-12 production, but only at high concentrations. Moreover, CSC, a selective A2a receptor antagonist, prevented the inhibitory effects of A1 and A3 receptor agonists. Therefore, it appears that the A1 and A3 receptor agonists express weak A2a receptor agonist effects at high concentrations. This is substantiated by previous studies showing that mouse peripheral lymphocytes (13, 14) and freshly isolated human monocytes express only functionally active A2 receptors (16, 19, 20, 22, 29). In contrast, cultured blood monocyte-derived macrophages and macrophage cell lines might express, in addition, functionally active A1 and/or A3 receptors (21, 29).
IL-12 is a central inducer of Th1 differentiation and serves as a bridge between innate and specific immunity, whereas IL-10 antagonizes the activities of IL-12 (1, 2, 3). The Th1/Th2 pattern is often regarded as a balance between Th1/Th2 cell cytokine activities, but our observations suggest that increased local concentrations of ADO may also contribute to Th1/Th2 balance. In addition to IL-12 inhibition, the present study also demonstrates that NECA and CGS-21680 dose-dependently potentiate the production of IL-10 in human whole blood ex vivo. This is in accordance with recent studies showing similar effects of ADO analogues on IL-10 production in vitro by human monocytes and in vivo in endotoxemic mice (23, 24). Interestingly, ADO appears to express identical modulatory effects on the balance of proinflammatory/anti-inflammatory cytokines such as PGE2 (26), catecholamines (30, 31) and histamine (27). In this process the increase of cAMP seems to be the common intracellular mechanism used by these endogenous mediators. Thus, conditions related to increased local concentrations of ADO, through inhibition of IL-12 and potentiation of IL-10 production from monocytes, may simultaneously mediate an inhibition of Th1 responses and a shift toward Th2 dominance.
One such condition is ischemia. During ischemia or hypoxia, local ADO concentrations increase to the micromolar range (11), whereas the secretion of TNF-α has been implicated in the pathogenesis of ischemia-reperfusion injury (32). Recent evidence indicates that ADO attenuates reperfusion injury following ischemia partly through inhibition of TNF-α production (33, 34). Our data suggest that during ischemia-reperfusion injury, increased concentrations of ADO may act to attenuate injury by inhibiting not only TNF-α release, but also IL-12 production. In this context, ADO might be a beneficial component of the normal host defense in mild ischemic foci. However, in more severe ischemic events, the systemic increase of ADO may have opposite, detrimental effects. Thus, major injury, serious burns, and brain injury often lead to severe immunosuppression causing increased susceptibility to infections (31, 35). Although it is still a matter of speculation, during major injury massive release of ADO may mediate, through inhibition of IL-12 production, part of the severe immunosuppression that occurs in these patients. The ADO-induced inhibition of IL-12 production may also be relevant to the immunosuppression observed in solid tumors, where hypoxic conditions cause accumulation of high concentrations of extracellular ADO that may contribute to inhibition of antitumor CTLs and NK activity (36).
Methotrexate and sulfasalazine are important second-line agents for the treatment of RA. Recent studies have indicated that these drugs enhance extracellular ADO concentrations (15, 18) and that ADO, via stimulation of A2 receptors, may be responsible in part for the anti-inflammatory effects of these drugs (15). Our results suggest that methotrexate, via release of ADO, may inhibit IL-12 production that may explain, at least in part, the beneficial effects of these drugs in RA patients.
In summary, we have shown that ADO analogues, via stimulation of A2a receptors and subsequent increase of cAMP, inhibit the production of IL-12 by activated human monocytes. Through this mechanism, increased concentrations of ADO may selectively suppress Th1 functions and skew the balance toward Th2 dominance. This may explain, at least in part, the anti-inflammatory properties of ADO and supports the rationale for development and use of selective A2a receptor agonists or antagonists in Th1- or Th2-mediated autoimmune processes, respectively.
Acknowledgments
We thank Dr. Michail V. Sitkovsky (Laboratory of Immunology, National Institute of Allergy and Infectious Diseases) and Dr. Thomas A. Fleisher (Immunology Service, Clinical Center, National Institutes of Health) for their helpful comments.
Footnotes
-
↵1 Address correspondence and reprint requests to Dr. Ilia Elenkov, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Building 10, Room 9N240, National Institutes of Health, 10 Center Drive, MSC 1820, Bethesda, MD 20892-1820. E-mail address: ElenkovI{at}mail.nih.gov
-
↵2 Abbreviations used in this paper: ADO, adenosine; RA, rheumatoid arthritis; SAC, Staphylococcus aureus Cowan strain 1; NECA, 5′-N-ethylcarboxamidoadenosine; N6-Benzyl-NECA, N6-Benzyl-5′-N-ethylcarboxamidoadenosine; IB-MECA, 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide CGS-21680, 2-p-(2-carbonylethyl)-phenylethylamino-5′-N-ethylcarboxamidoadenosine; CCPA, 2-Chloro-N6-cyclopentyladenosine; CSC, 8-(3-Chlorostyryl)caffeine; PKA, protein kinase A.
- Received October 5, 1999.
- Accepted October 19, 1999.
- Copyright © 2000 by The American Association of Immunologists
References
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