Stimulation of Eryptosis by Narasin

Narasin, a cationic ionophore [1] with antimicrobial activity [2,3,4,5] is fed to ruminant animals to improve feed efficiency [6,7]. It may accumulate in tissues of edible animals [8,9]. It is effective by permeabilizing cell membranes thus dissipating ion gradients of susceptible bacteria [6]. Narasin has been shown to inhibit NF-κB signaling [10]. Narasin fosters apoptosis of tumor cells [11] and has thus been considered for the therapy of malignancy [11].

In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis [12], the suicidal death of erythrocytes characterized by cell shrinkage [13] and cell membrane scrambling with phosphatidylserine translocation to the cell surface [12]. Eryptosis could be triggered by Ca²⁺ entry with increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). Stimulators of eryptosis further include ceramide [14], energy depletion [12], activated caspases [12,15,16], stimulated activity of casein kinase 1α [17], Janus-activated kinase JAK3 [18], protein kinase C [12], and p38 kinase [12,19], as well as impaired activity of AMP activated kinase AMPK [12], cGMP-dependent protein kinase [12], PAK2 kinase [12], and sorafenib/sunitinib sensitive kinases [12]. Eryptosis is stimulated by a wide variety of small molecules [12,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45]. Enhanced eryptosis is encountered in several clinical conditions, such as dehydration [33], hyperphosphatemia [43], chronic kidney disease (CKD) [25,46,47,48], hemolytic-uremic syndrome [49], diabetes [50], hepatic failure [51], malignancy [12,52], sepsis [53], sickle-cell disease [12,54], beta-thalassemia [12,54], Hb-C and G6PD-deficiency [12,54], as well as Wilsons disease [55].

The present study explored whether and how narasin triggers eryptosis. To this end, human erythrocytes from healthy volunteers were exposed to narasin and phosphatidylserine surface abundance, cell volume as well as [Ca²⁺]_i and ceramide abundance determined by flow cytometry.

Fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 x g for 20 min at 21 °C and the platelets and leukocytes-containing supernatant was disposed. Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO₄, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 5 glucose, 1 CaCl₂, at 37°C for 48 hours. Where indicated, erythrocytes were exposed to narasin (Sigma Aldrich, Hamburg, Germany) at the indicated concentrations.

After incubation under the respective experimental condition, a 150 µl cell suspension was washed in Ringer solution containing 5 mM CaCl₂ and then stained with Annexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 15 min under protection from light. The annexin V abundance at the erythrocyte surface was subsequently determined on a FACS Calibur (BD, Heidelberg, Germany). Annexin-V-binding was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. A marker (M1) was placed to set an arbitrary threshold between annexin-V-binding cells and control cells. The same threshold was used for untreated and narasin treated erythrocytes. A dot plot of forward scatter (FSC) vs. side scatter (SSC) was set to linear scale for both parameters. The threshold of forward scatter was set at the default value of “52”.

After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl₂ and 5 µM Fluo-3/AM. The cells were incubated at 37°C for 30 min and washed once in Ringer solution containing 5 mM CaCl₂. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µl Ringer solution and incubated at 37°C for 30 min. Then, Ca²⁺-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.

For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 hour at 37°C with 1 µg/ml anti ceramide antibody (clone MID 15B4, Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 minutes with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey's test as post-test and t test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.

The present study explored whether narasin is able to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface. Annexin-V-binding, as determined by flow cytometry, was taken as a measure of phosphatidylserine abundance at the erythrocyte surface. The erythrocytes were analysed following incubation for 48 hours in Ringer solution without or with narasin (1 - 25 ng/ml). As illustrated in Fig. 1, a 48 hours exposure to narasin increased the percentage of phosphatidylserine exposing erythrocytes, an effect reaching statistical significance at 10 ng/ml narasin concentration.

Forward scatter, as determined utilizing flow cytometry, was taken as a measure of erythrocyte volume. Again, measurements were made following a 48 hours incubation in Ringer solution without or with narasin (1 - 25 ng/ml). As shown in Fig. 2, low concentrations of narasin (1 ng/ml) significantly decreased erythrocyte forward scatter. However, forward scatter was not significantly modified by higher narasin concentrations (10 and 25 ng/ml). Thus, narasin exerted a dual effect on erythrocyte volume.

Fluo3 fluorescence was determined in order to estimate cytosolic Ca²⁺ activity ([Ca²⁺]_i). As illustrated in Fig. 3, a 48 hours exposure to 10 or 25 ng/ml narasin was followed by a significant increase of Fluo3 fluorescence, an observation pointing to an increase of [Ca²⁺]_i following narasin exposure.

In order to test whether the narasin-induced translocation of phosphatidylserine required entry of extracellular Ca²⁺, erythrocytes were incubated for 48 hours in the absence or presence of 25 ng/ml narasin in the presence or nominal absence of extracellular Ca²⁺. As illustrated in Fig. 4, removal of extracellular Ca²⁺ significantly blunted the effect of narasin on annexin-V-binding. However, narasin significantly increased the percentage of annexin-V-binding erythrocytes even in the absence of extracellular Ca²⁺. Thus, narasin-induced cell membrane scrambling was partially but not completely due to entry of extracellular Ca²⁺.

In search for an additional mechanism triggering eryptosis, ceramide abundance at the erythrocyte surface was quantified utilizing specific antibodies. As illustrated in Fig. 5, a 48 hours exposure to narasin (25 ng/ml) slightly but significantly increased the ceramide abundance at the erythrocyte surface.

The present study reveals a novel effect of narasin, i.e. the stimulation of suicidal erythrocyte death or eryptosis. Exposure of human erythrocytes to narasin results in decrease of forward scatter reflecting cell shrinkage and to increase of annexin-V-binding reflecting cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The concentrations required for the observed effect are within the range of plasma concentrations of treated poultry [56].

The effect of narasin on cell membrane scrambling was paralleled by and in part due to increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). Accordingly, the narasin induced cell membrane scrambling was blunted in the absence of extracellular Ca²⁺. However, narasin triggered cell membrane scrambling even in the absence of extracellular Ca²⁺, an observation pointing to involvement of additional mechanisms. Those mechanisms may include ceramide, which is slightly but significantly increased following narasin exposure.

An increase of [Ca²⁺]_i is further expected to decrease cell volume due to activation of Ca²⁺ sensitive K⁺ channels with subsequent cell shrinkage due to K⁺ exit, cell membrane hyperpolarization, Cl^- exit and thus cellular loss of KCl with water [13]. However, a decrease of cell volume was not observed at higher concentrations of narasin. Possibly the ionophore mediated ion entry outweighs the loss of KCl due to activation of Ca²⁺ sensitive K⁺ channels.

The physiological function of eryptosis is the clearance of defective erythrocytes from circulating blood prior to hemolysis [12]. Triggering of eryptosis avoids the release of hemoglobin, which would otherwise pass the glomerular filter of the kidney, precipitate in the acidic lumen of renal tubules and thus occlude nephrons [57]. In malaria, eryptosis allows the clearance of Plasmodium infected erythrocytes. Oxidative stress induced by the pathogen activates Ca²⁺-permeable erythrocyte cation channels in the infected host erythrocyte [12,58]. Eryptosis and subsequent clearance of infected erythrocytes are accelerated in patients carrying sickle-cell trait, beta-thalassemia-trait, Hb-C and G6PD-deficiency. The accelerated death of infected erythrocytes in those disorders counteracts parasitemia and thus protects against a severe course of malaria [12,59,60,61]. The enhanced eryptosis in iron deficiency [62], and following treatment with lead [62], chlorpromazine [63] or NO synthase inhibitors [63] similarly counteracts parasitemia. At least in theory, narasin could similarly enhance the susceptibility of Plasmodium infected erythrocytes to eryptosis.

Excessive eryptosis leads, however, to anemia [12]. Moreover, eryptosis may foster adherence of phosphatidylserine exposing erythrocytes to the vascular wall [64], stimulate blood clotting and trigger thrombosis [65,66,67]. Excessive eryptosis may thus compromise microcirculation [14,65,68,69,70,71].

In conclusion, narasin triggers cell membrane scrambling, an effect paralleled by and in part due to increase of cytosolic Ca²⁺ activity. Low concentrations of narasin further lead to cell shrinkage. Narasin thus triggers eryptosis, the suicidal death of erythrocytes.

The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch. The study was supported by the Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tuebingen University.

The authors of this manuscript state that they have no conflicts of interest to declare.