Nucleotidase) Up-Regulation ′ CD73

IFN-α Induced Adenosine Production on the Endothelium: A Mechanism Mediated by CD73 (Ecto-5′-Nucleotidase) Up-Regulation This information is current as Jussi Niemelä, Tiina Henttinen, Gennady G. Yegutkin, Laura of September 29, 2021. Airas, Anna-Maija Kujari, Pertti Rajala and Sirpa Jalkanen J Immunol 2004; 172:1646-1653; ; doi: 10.4049/jimmunol.172.3.1646 http://www.jimmunol.org/content/172/3/1646 Downloaded from References This article cites 41 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/172/3/1646.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 September 29, 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IFN-␣ Induced Adenosine Production on the Endothelium: (A Mechanism Mediated by CD73 (Ecto-5؅-Nucleotidase Up-Regulation1 Jussi Niemela¨,* Tiina Henttinen,* Gennady G. Yegutkin,* Laura Airas,* Anna-Maija Kujari,* Pertti Rajala,† and Sirpa Jalkanen2* CD73 (ecto-5؅-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-␣ produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-␣ treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to Downloaded from induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1␤, TNF-␣, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-␣. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-␣ is a relevant in vivo regulator of CD73 in the endothelial- leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflamma- http://www.jimmunol.org/ tion via CD73-dependent adenosine production. The Journal of Immunology, 2004, 172: 1646–1653. he interaction between lymphocytes and endothelial cells cially interesting in light of the fact that the cDNA and protein is a multistep process. To be able to penetrate the vessel structure are very similar on both cell types (7). T wall and to reach the target site, circulating cells use a Adenosine, a purine nucleoside product of CD73 enzyme activ- very finely regulated set of adhesion molecules. Enhanced adhe- ity, has a role in many physiological and pathological events. It sion to endothelium and subsequent transmigration of recirculating binds to specific receptors on the cell surface. To date, four dif- leukocytes through the endothelial lining of vessel wall into the ferent subtypes of G protein-coupled adenosine receptors, A1R, by guest on September 29, 2021 tissue are characteristic for inflammation. Moreover, the release of A2aR, A2bR, and A3R, have been cloned (9). Ecto-5Ј-nucleoti- pro- and anti-inflammatory cytokines in a high extent takes place dase activity is shown to be an important mediator of the anti- at sites of inflammation. Those cytokines are potent regulators of inflammatory effect of methotrexate and sulfasalazine in vitro and the expression of adhesion molecules (1–3). in vivo in the murine air pouch inflammation model through in- Ј Ecto-5 -nucleotidase (CD73) is a 70-kDa GPI-anchored cell sur- creasing extracellular adenosine levels (10). Adenosine, by bind- face molecule with ecto-enzymatic activity. It is abundantly ex- ing to A1 and A2 receptors, regulates pathological consequences pressed on the vascular endothelium and at a low level on certain of inflammation by controlling leukocyte binding to endothelium subpopulations of human lymphocytes. It is part of the purine sal- (11) and acts as an anti-inflammatory agent by binding to A2 and Ј vage pathway by degrading nucleoside-5 -monophosphates (AMP A3 receptors, through the inhibition of neutrophil degranulation and IMP) into nucleosides such as adenosine and inosine (4, 5). (12). Adenosine also decreases eosinophil migration through acti- CD73 has also been suggested to mediate homing of skin-infil- vation of A3 receptor (13). Adenosine, converted from neutrophil- trating lymphocytes in vivo (6). Triggering of CD73 on the surface derived AMP, leads to increased endothelial barrier function by of lymphocytes, but not on endothelial cells, results in the shed- endothelial A2bR activation. This promoting effect of AMP is ding of the CD73 and increased adhesion of lymphocytes to en- CD73 mediated and is followed by an increase in intracellular dothelium via LFA-1 clustering (7, 8). This phenomenon is espe- cAMP (14). Recently, a critical role for A2a receptor has been shown in decreasing systemic and tissue-specific inflammatory re- *MediCity Research Laboratory and Department of Medical Microbiology, Turku sponses in vivo (15). University, Turku and National Public Health Institute, Turku, Finland; and †Depart- ment of Surgery, Turku University Central Hospital, Turku, Finland To date, practically nothing is known about the regulation of Received for publication May 28, 2003. Accepted for publication November endothelial CD73 expression and function. However, in inflam- 20, 2003. mation there may be some inducers secreted that in vivo specifi- The costs of publication of this article were defrayed in part by the payment of page cally control endothelial CD73 expression. This hypothesis was charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. suggested by a previous finding, that CD73 is up-regulated in in- flamed skin (6). 1 This work was supported by grants from the Finnish Academy, the Sigrid Juselius Foundation, the Finnish Cancer Union, the Ida Montin Foundation, the Paulo Foun- As adenosine, having an anti-inflammatory and cell-protective dation, the Research and Science Foundation of Farmos, the Finnish Cultural Foun- effect, plays an important role in controlling the extent and con- dation, and the Turku Graduate School of Biomedical Sciences. sequences of inflammation, this work was designed to identify the 2 Address correspondence and reprint requests to Dr. Sirpa Jalkanen, MediCity Re- search Laboratory, Turku University, Tykisto¨katu 6A, FIN-20520 Turku, Finland. factors responsible for the regulation of CD73 expression as well E-mail address: sirpa.jalkanen@utu.fi as ecto-5Ј-nucleotidase-mediated adenosine production. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 1647 Materials and Methods Table I. Regulators used to induce CD73 expression Cells, Abs, and reagents Inducer Concentration Used Incubation Time HUVEC (3) were isolated using a method modified from Jaffe et al. (16), and were cultured on gelatin-coated cell culture flasks in complete medium, Rapidly acting as described earlier (7). Human PBL from healthy volunteers were isolated FMLP 10Ϫ9 and 10Ϫ7 M 5 min and 2 h using Ficoll-Hypaque (Histopague-1077; Pharmacia, Uppsala, Sweden). Dibutyryl cAMP 0.5 and 5 ␮g/ml 5 min and 2 h PBL and U266B1 cell line, a gift from J.-Y. Bonnefoy (Glaxo Institute for Histamine 1 and 10 ␮g/ml 5 min and 2 h Molecular Biology, Geneva, Switzerland), was cultured in RPMI 1640 PMA 1 and 10 ng/ml 5 min and 2 h medium containing 10% FCS, 4 mM L-glutamine, 100 U/ml penicillin, and Slowly acting 100 ␮g/ml streptomycin. Anti-CD73 mAb 4G4 (mouse IgG1), anti- IL-1␤ 10 and 100 U/ml 4, 20 h ICAM-1 mAb 5C3 (IgG1) (17), anti-CD31 mAb 2C8 (IgG1), and mAb IL-4 10 and 100 U/ml 4, 20 h 3G6 (mouse IgG1) against chicken T cells as a negative control Ab were 100 and 500 U/ml 48 h used. ␣,␤ Methyleneadenosine 5Ј-diphosphate (AMPCP)3 and AMP were TNF-␣ 10 and 200 U/ml 4, 20 h from Sigma-Aldrich (St. Louis, MO). 200 and 1000 U/ml 48 h Inductions and immunofluorescence stainings LPS 10 and 100 ng/ml 4, 20 h 100 and 500 ng/ml 48 h IL-1␤, IL-4, TNF-␣, and IFN-␥ (Genzyme, Cambridge, MA); IFN-␣ IFN-␣ 1, 5, 10, 50, 100, 200, 72 h (Wellferon; The Wellcome Foundation, London, U.K.); LPS from Esche- 500, and 2000 U/ml richia coli serotype O:55 (Difco Laboratories, Detroit, MI); and dibutyryl 1000 U/ml 4, 12, 20, 24, 48, cAMP, PMA, thrombin from human plasma, histamine, and FMLP from 60, 72, and 96 h Sigma-Aldrich were used for inductions, as indicated in Table I. For every IFN-␥ 50 and 200 U/ml 4 and 20 h Downloaded from time point, a control flask was incubated without inducers. 1000 U/ml 12, 20, 24, 48, 60, and 72 h Three different protocols were used for immunofluorescence stainings 1) To study the effect of a panel of different inducers on surface expression of CD73 immunofluorescence analyses were performed, as reported before using TaqMan Universal PCR Master Mix and ABI PRISM 7700 Sequence (17).

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