Artikel
Membrane-associated AMPD2, a novel regulator in shifting the balance between extracellular ATP and adenosine?
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Autoren
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Lisa Ehlers
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
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Aditi Kuppe
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
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Alexandra Damerau
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
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Yuling Chen
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
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Cindy Strehl
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
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Marieluise Kirchner
- Max Delbrück Center for Molecular Medicine, BIH Core Facility Proteomics, Berlin
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Frank Buttgereit
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
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Timo Gaber
- Charité – Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Rheumatologie und Klinische Immunologie, Berlin
| Veröffentlicht: | 5. Februar 2019 |
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Gliederung
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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 and adenosine by the action of the ectonucleotidases CD39 and CD73, causing a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine. Intracellularly, AMPD2 executes AMP deamination to IMP thereby reducing adenosine formation. 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. Therefore, we analyzed surface AMPD2 expression and its modulation on distinct cell lines and primary 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 was analyzed on PBMCs and isolated monocytes. Association of surface AMPD2 and cell death was visualized using annexin V and 7-AAD staining and examined by flow cytometry. In addition, expression of AMPD2 was analyzed 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 show 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 monocytes after 24h whereas AMPD2 surface expression is reduced in T cells treated with LPS and PHA respectively, (iii) that LPS decreases CD73 expression on monocytes, and (iv) that all cell lines analyzed 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.
Conclusion: 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.
