Ligand-Activation of the Adenosine A2a Receptors Inhibits IL-12 Production by Human Monocytes This information is current as Amrey A. Link, Tomoshige Kino, James A. Worth, Jennifer of October 1, 2021. L. McGuire, Marianna L. Crane, George P. Chrousos, Ronald L. Wilder and Ilia J. Elenkov J Immunol 2000; 164:436-442; ; doi: 10.4049/jimmunol.164.1.436 http://www.jimmunol.org/content/164/1/436 Downloaded from References This article cites 36 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/164/1/436.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Ligand-Activation of the Adenosine A2a Receptors Inhibits IL-12 Production by Human Monocytes Amrey A. Link,* Tomoshige Kino,* James A. Worth,* Jennifer L. McGuire,* Marianna L. Crane,† George P. Chrousos,* Ronald L. Wilder,† and Ilia J. Elenkov1*† 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 inhib- ited, 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-b-D-ribofuranuronamide expressed only weak Downloaded from inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differ- ential 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 in- http://www.jimmunol.org/ hibitor, 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. The Journal of Immunology, 2000, 164: 436–442. ellular and humoral immunity and their effector cells diabetic mice (5) and in type II collagen-induced arthritis in are regulated by APCs, the Th cell subtypes Th1 and DBA/1 mice (6). In addition, stimulation of IL-12 secretion by Th2, and by their secreted soluble messengers (1, 2). Th1 microbial products was shown to be the crucial factor for the pro- C by guest on October 1, 2021 cells primarily secrete IFN-g, IL-2, and TNF-b, which promote liferation and differentiation of pathogenic autoreactive Th1 effec- cellular immunity, whereas Th2 cells secrete a different set of cy- tor cells in experimental allergic encephalomyelitis (7). tokines, primarily IL-4, IL-10, and IL-13, which promote humoral Adenosine (ADO),2 together with ADP and ATP, belongs to a immunity (1, 2). class of endogenous purine nucleosides produced by many cells IL-12 is a 75-kDa heterodimeric cytokine composed of p35 and during normal metabolic activity (8–10). These ubiquitous mole- p40 chains; p35 is constitutively expressed in most cells, whereas cules are utilized in selective extracellular signaling (8–10). For p40 is up-regulated in monocytes and macrophages in response to example, ADO appears to be an important endogenous regulator of infection or appropriate activating substances such as LPS (3). coronary blood flow (8). Postganglionic sympathetic nerve termi- IL-12 is a central inducer of Th1 responses and cell-mediated im- nals also release ATP that is rapidly degraded to ADO, which munity. First, it induces IFN-g production from NK and T cells, induces vasodilatation mediated by A2 receptors (10). Local ex- which contributes to phagocytic cell activation and inflammation. tracellular ADO levels increase dramatically during ischemia and Second, it favors Th1 cell differentiation and proliferation (3). hypoxia, possibly providing cytoprotection and increased blood IL-12 is essential for the clearance of certain intracellular patho- flow to ischemic tissues (11). Inflammation and tissue injury rep- gens such as Mycobacterium tuberculosis (4). However, excessive resent pathologic states that are also associated with enhanced ex- production of IL-12 may be responsible for the proinflammatory tracelluar ADO concentrations. activities and the tissue damage typical of organ-specific autoim- One of the first clinical observations linking ADO to the im- munity, as seen in insulin-dependent diabetes mellitus in nonobese mune system was made in patients with SCID. These patients lack the enzyme adenosine deaminase, i.e., they are unable to catabo- lize ADO (12). This abnormality results in highly elevated plasma *Developmental Endocrinology Branch, National Institute of Child Health and Hu- man Development, and †Arthritis and Rheumatism Branch, National Institute of Ar- levels of ADO and consequent impairment of cellular immunity, thritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Be- marked by recurrent infections. The immunosuppression observed thesda, MD 20892 Received for publication October 5, 1999. Accepted for publication October 19, 1999. The costs of publication of this article were defrayed in part by the payment of page 2 Abbreviations used in this paper: ADO, adenosine; RA, rheumatoid arthritis; SAC, charges. This article must therefore be hereby marked advertisement in accordance Staphylococcus aureus Cowan strain 1; NECA, 59-N-ethylcarboxamidoadenosine; N 6- with 18 U.S.C. Section 1734 solely to indicate this fact. Benzyl-NECA, N 6-Benzyl-59-N-ethylcarboxamidoadenosine; IB-MECA, 1-deoxy-1-[6- 1 Address correspondence and reprint requests to Dr. Ilia Elenkov, Arthritis and Rheu- [[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-b-D-ribofuranuronamide CGS- matism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, 21680, 2-p-(2-carbonylethyl)-phenylethylamino-59-N-ethylcarboxamidoadenosine; Building 10, Room 9N240, National Institutes of Health, 10 Center Drive, MSC 1820, CCPA, 2-Chloro-N6-cyclopentyladenosine; CSC, 8-(3-Chlorostyryl)caffeine; PKA, pro- Bethesda, MD 20892-1820. E-mail address: [email protected] tein kinase A. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 437 in SCID is explained by the direct lymphotoxicity of ADO catabo- Monocyte elutriation and culture lites and by the strong extracellular ADO-induced inhibition of the Human monocytes were obtained from healthy volunteers of the National TCR-triggered proliferation and T cell effector functions mediated Institutes of Health, Department of Transfusion Medicine, Cell Processing by A2a receptor signaling (13, 14). More recently, it has been Section. Mononuclear cells were isolated on lymphocyte separation me- demonstrated that ADO also exerts diverse anti-inflammatory ef- dium and the monocytes were purified by counterflow centrifugal elutria- 3 5 fects, mediated mainly by A2 receptors, including diminished leu- tion as previously described (25). The monocytes were cultured at 5 10 cells/ml. All cultures were set up in RPMI 1640 medium supplemented kocyte accumulation, inhibition of C2 production, and reduction of with 15% FCS, 1% glutamine, and 50 mg/ml gentamicin to yield a final the superoxide anion generation (15–17). Therefore, the regulation volume of 1.5 ml. Cytokine production was induced by LPS (1 mg/ml), and of ADO receptors is recognized as having significant therapeutic all cultures were incubated in 5% CO2 at 37°C for 18 h. In several exper- potential (9). iments, anti-IL-10 Abs were added at a concentration of 10 mg/ml. Ac- cording to the manufacturer’s instruction, this concentration gives a 50% Recent evidence suggests that the anti-inflammatory effects of neutralizing dose in the presence of 5 ng/ml of rhIL-10. methotrexate and sulfasalzine, currently the most commonly pre- scribed and effective second-line agents for the treatment of rheu- Cytokine assays matoid arthritis (RA), are mediated in part by increases in the extracellular concentrations of ADO (15, 18). Because excessive IL-12 (p40 and p70) and IL-10 were measured using ELISAs employing the multiple-Ab sandwich principle (Quantikine, R&D Systems). These production of IL-12 appears to play an important role in RA, the assays specifically detect human IL-12 p70 (the biologically active het- question arises: does ADO suppress IL-12 production? This may erodimer), p40, and human IL-10, respectively.