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. 2019 May 10;10:1041. doi: 10.3389/fimmu.2019.01041

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Copyright © 2019 Ulivieri, De Tommaso, Finetti, Ortensi, Pelicci, D'Elios, Ballerini and Baldari.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Rai dampens extracellular ATP-degrading enzyme activity in astrocytes. (A) ATP (eATP) quantification in culture supernatants from Rai^+/+ (Astro WT) and Rai^−/− (Astro Rai^−/−) astrocytes stimulated for 5 h with IFNγ (10 ng/ml) or IL-17 (50 ng/ml) or left untreated (-). Data are presented as mean ± SD of relative luciferase units (RLU) in supernatants from Rai^−/− astrocytes vs. Rai^+/+ astrocytes. Data have been normalized to the mean RLU value of Rai^+/+ astrocytes (n = 5). (B) Total ATP (tATP) content in unstimulated Rai^+/+ (Astro WT) and Rai^−/− (Astro Rai^−/−) astrocytes. (C) Flow cytometric analysis of surface CD73 and CD39 in Rai^+/+ (Astro WT) and Rai^−/− (Astro Rai^−/−) astrocytes stimulated for 5 h with IFNγ (10 ng/ml), IL-17 (50 ng/ml) or left untreated (-). Data are presented as mean ± SD of mean fluorescence intensity (MFI) (n = 4). (D) Quantification of enzymatic activities of extracellular ATP-degrading enzymes in Rai^+/+ (Astro WT) and Rai^−/− (Astro Rai^−/−) astrocytes stimulated with IL-17 or IFNγ for 5 h or left untreated (-), then depleted of their culture supernatant and incubated with 1 mM ATP. Data are presented as mean fold change ± SD of specific enzymatic activities (nmol free phosphate/mg protein/min) in Rai^+/+ astrocytes and Rai^−/− astrocytes, with unstimulated Rai^+/+ astrocytes taken as 1 (n = 3). (E) Quantification of adenosine in culture supernatants of astrocytes treated as in D. Data are presented as mean ± SD of adenosine concentration (μM) (n = 3). (F) Immunoblot analysis with anti-Rai or anti-CD39 antibodies of RanBPM-specific immunoprecipitates from total cell lysates of Rai^+/+ and Rai^−/− astrocytes treated with IFNγ (10 ng/ml) for 15 min (n = 2). The quantification by laser densitometry of the levels of each of the proteins normalized to the level of RanBPM in each sample is shown (n = 2). 2-Way ANOVA and Mann–Whitney test ^****p < 0.0001, ^**p < 0.01, ^*p < 0.05.