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Session 1: Cell death Poster 15
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ATP regulated Apoptosis versus Autophagy in Cultured Dendritic Cells.
Sarah Flacke 1 , Patricia Schilling 1 , Fengguang Liu 1 , Karl J. Foehr 1 , Manfred Weiss, Paul
Walther 2 , E. Marion Schneider 1
1 Sektion Experimentelle Anaesthesiologie, University Hospital Ulm, Steinhoevelstr. 9, 89075
Ulm, Germany; 2 Electron Microscopy Facility, Ulm University, Ulm, Germany
Cell stress may lead to the release of nucleotides in many tissues. Specifically, fluid shear, cell
stretching, hypoxia, osmotic swelling, but also temperature-related stimuli induce ATP release in
addition to other nucleotides. Extracellular nucleotides bind to cell surface P2 receptors, either P2X,
which are ATP-gated nonselective cation channels, or P2Y, which are G-protein-coupled receptors.
There are seven subtypes of P2X (P2X1–7) and eight subtypes of P2Y (P2Y1, -2, -4, -6, and -11–14)
receptors currently identified in mammals. A major effect occurs by ATP following stimulation of the
P2RX7 receptor leading to extensive cell blebbing in addition to pore formation, facilitating the
transport of molecules as large as 900 kD. Amongst cells of the hematopoietic system, macrophages
and dendritic cells express P2RX7 receptors at high density. The expression of P2RX7 as well as its
functional activity can be further increased by in vitro culture in the absence of exogenous growth
factors and cytokines. Using cell biology methods, patch clamping, caspase 3/7 and caspase 8
measurements in lysates, and or LC-3 staining by flow cytometry, we determined the effect of
extracellular ATP on cultured dendritic cells from patients with macrophage activation syndromes
(MAS). In contrast to healthy donors, MAS patients were unique by their increased precursor
frequencies of P2RX7 high expressing dendritic cells in peripheral blood. We found two types of
ATP- responses in selected donors: One which induced apoptosis following ATP-stimulation and
another mediated increased LC-3 expression and autophagy by the same ATP stimulation protocol.
These differential results corresponded to a donor-specifically smaller (about 10pA/pF) or larger
(about 100pA/pF) electrophysiological response as determined by patch clamping. Pyrosequencing
was applied to study functional single nucleotide polymorphisms of the P2RX7 receptor in different
donors. Out of 5 SNPs investigated*, the P2RX7 signal intensity as well as pro- vs. anti-apoptosis and
the corresponding induction of autophagy in these dendritic cells, was largely related to a 1513A!C
polymorphism, responsible for a Glu496!Ala exchange in the intracellular C-terminus of the P2RX7
receptor. We therefore conclude that functional SNPs of the P2RX7 receptor are responsible for
dichotomous stress-induced and P2RX7-mediated response leading to either apoptosis or increased
survival of immature dendritic cells. Moreover, this observation may explain a differential traumainduced
stress response in vivo.
*Geistlinger et al. Clin Chim Acta, in press.
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