Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca 2+ -activity ([Ca 2+ ] i ), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from

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... g, cell volume from forward scatter, [Ca 2+ ] i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca 2+ ] i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca 2+ ] i , increase of ceramide abundance and caspase activation. be stimulated following activation of casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, and/or p38 kinase [2] . Eryptosis is inhibited by AMP activated kinase AMPK, cGMPdependent protein kinase, and PAK2 kinase [2] . Eryptosis is stimulated by a wide variety of small molecules [2, . Moreover, eryptosis is enhanced in elderly individuals [64] . It is sensitive to erythrocyte age [65] and enhanced eryptosis is observed following erythrocyte storage [66, 67] . Eryptotic erythrocytes are bound to and subsequently engulfed by macrophages and thus rapidly cleared from circulating blood [2, 68] . Accordingly, excessive eryptosis may lead to anemia [2] . Moreover, eryptotic erythrocytes may bind to endothelial cells of the vascular wall [69], trigger blood clotting and induce thrombosis [70] [71] [72] , thus interfering with microcirculation [5, 70, [73] [74] [75] [76] . A variety of clinical conditions are paralleled by stimulated eryptosis [2-4, 77-84] including fever [85] . Fever is a common complication of appendicitis [86] , which is paralleled by an increase of C-reactive protein abundance in plasma [87, 88] . Enhanced CRP plasma levels are valuable markers of inflammation [89, 90] . C-reactive protein contributes to the host reaction against pathogens and participates in the orchestration of inflammation by interaction with endothelial cells, endothelial progenitor cells, leukocytes and platelets [90] . The inflammatory response is accomplished by pentameric C-reactive protein (pCRP) [89] . C-reactive protein has been shown to trigger suicidal death or apoptosis of nucleated cells [91] and could thus, at least in theory, similarly stimulate eryptosis. The present study addressed the impact of C-reactive protein on eryptosis. To this end, erythrocytes drawn from healthy individuals were exposed to C-reactive protein and [Ca 2+ ] i , ceramide abundance and caspase activity were determined. Moreover, blood was drawn from patients with acute appendicitis or erythrocytes from healthy individuals exposed to plasma from appendicitis patients and eryptotic erythrocytes identified by determination of phosphatidylserine exposure. Materials and Methods Patients, erythrocytes and treatments Blood was drawn from untreated patients diagnosed with acute appendicitis (11 ♀, 9 ♂, age range 22-76 years) and healthy volunteers (10 ♀, 10 ♂, age 22-69 years). The patients were recruited 2012 and 2013 from the Department of General, Visceral and Transplant Surgery, Tübingen, Germany. The study was approved by the ethics committee of the University of Tübingen (184/2003V). Both, patients and healthy volunteers provided written informed consent. Measurements were made in freshly isolated erythrocytes or in erythrocytes (Oblood group) from healthy young individuals incubated in vitro with fresh plasma from either patients or healthy volunteers. Alternatively, fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. To isolate erythrocytes, blood was centrifuged at 120 g for 20 min at 23 °C, the platelets and leukocytes-containing supernatant was disposed, and the erythrocyte pellet washed once with Ringer solution. Erythrocytes were incubated in vitro for 24 hours at a hematocrit of 0.4% in plasma from patients or healthy volunteers or in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, and 1 CaCl 2 ; the pH was adjusted to 7.4 and the temperature kept at 37°C. The low hematocrit was chosen to minimize mutual erythrocyte interaction. Where indicated, erythrocytes were exposed to C-reactive protein (Sigma Aldrich, Hamburg, Germany) at the indicated concentrations. Annexin-V-binding and forward scatter After incubation under the respective experimental conditions, 150 µl cell suspension was washed in Ringer solution containing 5 mM CaCl 2 and then stained with Annexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany).