Session Type: Poster Session (Sunday)
Session Time: 9:00AM-11:00AM
Background/Purpose: Adenosine and its nucleotides represent crucial immunomodulators in the extracellular environment. ATP and ADP are released from stressed cells in states of inflammation, whereas adenosine serves as a key anti-inflammatory mediator which signals via multiple receptors on immune cells. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thereby promoting an anti-inflammatory milieu induced by the “adenosine halo”. Great importance has been attributed to these enzymes in the pathogenesis of autoimmune diseases such as RA and as targets in cancer therapy. AMPD2 mediates AMP deamination to IMP, consequently reducing adenosine formation. Here, we postulate that (i) this pathway is also present on the cell surface of immune cells, and (ii) its predominance may lead to an increased state of inflammation as found in chronic inflammatory diseases (Fig. 1).
Therefore, we analyzed surface AMPD2 expression and its modulation on distinct cell lines and primary immune cells.
Methods: Firstly, AMPD2 surface expression was verified by immunoprecipitation from membrane fractions isolated from cell lines (HEK293 and HMEC1) and CD14+ monocytes analyzed by immunoblot and mass spectrometry. In addition, surface biotinylation of the aforementioned cells was performed. Also, AMPD2 surface expression was evaluated by flow cytometry, analyzing both cell lines (HEK293, HMEC1, THP1, and Jurkat) and primary human immune cells.
Secondly, co-expression of surface AMPD2, CD39 and CD73 on PBMCs was analyzed by flow cytometry directly after isolation as well as after a 24h culture period. Moreover, surface expression was assessed after immunostimulation and Golgi transport inhibition.
Results: AMPD2 surface expression was confirmed by immunoblot and mass spectrometry of (i) precipitated AMPD2 from membrane fractions and (ii) biotinylated surface molecules in HEK293 and HMEC1 as well as CD14+ monocytes. Surface expression was reduced after AMPD2 knockdown in HEK293. AMPD2 was detected on the surface of all examined cell lines, human T cells and monocytes from healthy donors and RA patients by flow cytometric analysis. Flow cytometry revealed a stable surface expression of AMPD2, CD39 and CD73 on lymphocytes after immunostimulation. Golgi transport inhibition slightly reduced AMPD2 surface expression (p=0.02), while expression of CD39 and CD73 remained unaffected. However, 24h incubation with substances agonizing TLRs 1, 2, 4, 5, 6, 7, and 8 respectively strongly enhanced the surface expression of AMPD2 (p< 0.01) and CD39 (p< 0.05) on monocytes, while this effect was attenuated by concomitant incubation with dexamethasone. Agonists of TLRs 3 and 9 did not affect AMPD2 surface expression. CD73 expression was not influenced by immunostimulation. Golgi transport inhibition significantly decreased monocytic AMPD2 surface expression (p< 0.001).
Conclusion: We demonstrate AMPD2 surface expression on immune cells for the first time and thereby reveal a novel regulator of the extracellular ATP-adenosine balance. The extracellular conversion of AMP into IMP may constitute a shunt-like mechanism adding to the CD39-CD73 system controlling immunomodulation.
To cite this abstract in AMA style:Ehlers L, Kuppe A, Strehl C, Kirchner M, Buttgereit F, Gaber T. AMP Deaminase 2 Surface Expression Counteracting CD73-Driven Generation of Anti-Inflammatory Extracellular Adenosine [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/amp-deaminase-2-surface-expression-counteracting-cd73-driven-generation-of-anti-inflammatory-extracellular-adenosine/. Accessed November 28, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/amp-deaminase-2-surface-expression-counteracting-cd73-driven-generation-of-anti-inflammatory-extracellular-adenosine/