SAT0038 Membrane-associated ampd2, a novel regulator in shifting the balance between extracellular atp and adenosine?. (12th June 2018)

• Record Type: Journal Article
• Authors: Ehlers, L.; Kuppe, A.; Damerau, A.; Chen, Y.; Buttgereit, F.; Gaber, T.
• Is Part Of: Annals of the rheumatic diseases. Volume 77(2018)Supplement 2
• Page Start: 883
• Page End: 884
• Publication Date: 2018-06-12
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2018-eular.6608
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Background: Extracellular ATP and adenosine are potent immunomodulatory molecules that accumulate in states of inflammation. ATP/ADP are released from damaged or stressed cells and sequentially catabolized to AMP which is then catabolized to adenosine by the action of the ectonucleotidases CD39 and CD73, which drives a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine. Intracellularly, AMPD2 which encodes one of three known AMP deaminase homologs executes AMP deamination to IMP thereby reducing the formation of adenosine. Here, we postulate that this mode of action is also present on the cell surface of immune cells, which may lead to an increased state of inflammation such as found in chronic inflammatory diseases. Objectives: Therefore, we analysed surface AMPD2 expression and its modulation on distinct human cell lines and primary human immune cells. Methods: To this end, surface AMPD2 expression was evaluated on cell lines (THP1, Jurkat, HMEC1, and HEK293), human PBMCs and isolated monocytes by flow cytometry. Moreover, co-expression of surface AMPD2, CD73 and CD39 has been analysed on PBMCs and isolated monocytes. Association of surface AMPD2 and cell death was visualised using annexin V and 7-AAD staining and examined by flow cytometry. In addition, expression of AMPD2 was analysed by immunoblot of precipitated AMPD2 from membrane fractions and by mass spectrometry after precipitation from membrane fractions and from biotinylated surface-molecules using the surface AMPD2 positive cell line HEK293. Results: Here, we demonstrate that (i) surface AMPD2 is present on T cells and monocytes in PBMCs from healthy donors, (ii) that LPS enhances surface expression of AMPD2 in PBMCs on both cell populations after 24 hour, (iii) that LPS also enhances surface expression of AMPD2 in isolated monocytes, (iv) that surface AMPD2 does not correlate to the expression of the ectonucleotidases CD39 and CD73 and (v) that all cell lines analysed are capable of expressing surface AMPD2. Using HEK293, surface AMPD2 expression was reduced after Golgi transport inhibition. AMPD2 surface expression was not accompanied by enhanced cell death. Expression of AMPD2 could be confirmed in membrane fractions of HEK293 using immunoblot of precipitated AMPD2 and mass spectrometry, respectively. Conclusions: Here, we demonstrate for the first-time surface expression of AMPD2 on immune cells enabling these cells to extracellularly convert AMP into IMP constituting a shunt-like mechanism to control the levels of adenosine and extracellular ATP formed from adenine nucleotides thereby controlling immunomodulation. Disclosure of Interest: None declared