Background/Aims: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca2+ entry and increased ceramide abundance.

Keywords:
Phosphatidylserine, Cell volume, Eryptosis, Oxidative stress, Calcium

Carnosic acid, a phenolic abietane diterpene component of rosemary and sage [1,2,3,4,5,6,7], has been shown to exert anti-oxidative [1,2,8,9,10,11,12,13,14,15,16,17,18], anti-inflammatory [9,19,20,21,22,23], anti-adipogenic [2,22,23,24,25,26,27,28,29,30,31], anti-diabetic [24,27,29,32], anti-fatty liver [33], anti-tumor [6,20,32,34,35,36,37,38,39,40], anti-angiogenic [20,41], anti-microbial [1,6,32,42,43], pro-apoptotic [19,26,37,44,45,46,47], pro-autophagic [48], anti-apoptotic [5,13,14,16], gastroprotective [15], and neuroprotective [5,8,9,11,16,32,49,50,51,52,53,54,55] effects. Cellular mechanisms involved in the effects of carnosic acid include up-regulation of Nuclear factor (erythroid-derived 2)-like 2 (Nfr2) activity [8,13,55,56], preservation of mitochondrial respiratory function [11,56], mitochondrial depolarization [47], cytochrome-c release [36], increase in Bax:Bcl-2 ratio [36], caspase activation [36,37], up-regulation of NAD+-dependent deacetylase SIRT1 [2], glutathione S-transferase [8,57], death receptor (DR)5 [19], Bcl-2 interacting mediator of cell death (Bim) [19] and p53 up-regulated modulator of apoptosis (PUMA) [19] expression, down-regulation of c-FLIP and Bcl-2 expression [19] as well as stimulation of PGE2 synthesis [15]. Signaling involved in the effect of carnosic acid further includes phosphatidylinositol 3-kinase (PI3K)/Akt [26,46,47,48,49], nuclear factor-kappa B (NF-κB) [21,49], and p38 kinase [8,16,45,57].

To the best of our knowledge, nothing is known about effects of carnosic acid on survival of erythrocytes, cells lacking mitochondria and nuclei. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis [58], the suicidal erythrocyte death characterized by cell shrinkage [59] and cell membrane scrambling with phosphatidylserine translocation to the cell surface [58]. Eryptosis may be triggered by activation of Ca2+ permeable unselective cation channels with subsequent Ca2+ entry and increase of cytosolic Ca2+ activity ([Ca2+]i) [58]. Mechanisms fostering eryptosis further include ceramide [60], energy depletion [58], activated caspases [58,61,62], activation of casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, and p38 kinase [58], as well as inhibition or lack of AMP activated kinase AMPK, cGMP-dependent protein kinase, PAK2 kinase, and sorafenib/sunitinib sensitive kinases [58]. Eryptosis could be stimulated by a wide variety of xenobiotics [58,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87]. Moreover, enhanced eryptosis is observed in several clinical conditions, including dehydration [76], hyperphosphatemia [86], chronic kidney disease (CKD) [68,88,89,90], hemolytic-uremic syndrome [91], diabetes [92], hepatic failure [93], malignancy [58], sepsis [94], Sickle-cell disease [58], beta-thalassemia [58], Hb-C deficiency, G6PD-deficiency [58] and Wilsons disease [95].

The present study explored whether and how carnosic acid could trigger or inhibit eryptosis. To this end, human erythrocytes from healthy volunteers were treated with carnosic acid and phosphatidylserine surface abundance, cell volume, [Ca2+]i, ROS formation, and ceramide abundance determined by flow cytometry.

Erythrocytes, solutions and chemicals

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 carnosic acid (Sigma Aldrich, Hamburg, Germany) at the indicated concentrations.

Annexin-V-binding and forward scatter

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 carnosic acid 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”.

Intracellular Ca2+

After incubation, a 150 µl cell suspension was centrifuged at 1600 rpm for 3 mins and, after trashing the supernatant, the erythrocytes were stained with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM 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.

Reactive oxidant species (ROS)

Oxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 150 µl suspension of erythrocytes was centrifuged at 1600 rpm for 3 min and, after trashing the supernatant, the erythrocytes were stained with DCFDA (Sigma, Schnelldorf, Germany) in Ringer solution containing DCFDA at a final concentration of 10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and then washed in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 µl Ringer solution, and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).

Ceramide abundance

For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, a 100 µl cell suspension was centrifuged at 1600 rpm for 3 min, and, after trashing the supernatant, the erythrocytes 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. After two washing steps with PBS-BSA, cells were stained for 30 min with polyclonal fluorescein- isothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody (BD Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. Samples were then analyzed by flow cytometric analysis at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Hemolysis

In order to determine hemolysis, the samples were centrifuged (3 mins at 1600 rpm, room temperature) after incubation under the respective experimental conditions and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100 % hemolysis. Hemolysis is expressed in % in order to allow comparison with % annexin V binding cells.

Statistics

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 the influence of carnosic acid on eryptosis, the suicidal erythrocyte death characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. In order to identify phosphatidylserine exposing erythrocytes, phosphatidylserine was stained with FITC-labelled annexin and annexin-V-binding quantified by flow cytometry. The erythrocytes were analyzed following incubation for 48 hours in Ringer solution without or with carnosic acid (1-10 µg/ml). As illustrated in Fig. 1, a 48 hours exposure to carnosic acid increased the percentage of phosphatidylserine exposing erythrocytes, an effect reaching statistical significance at 2.5 µg/ml carnosic acid.

The suicidal erythrocyte death could involve hemolysis, a cell death distinct from eryptosis. In order to estimate the effect of carnosic acid on hemolysis, the hemoglobin concentration in the supernatant was determined. As a result, a 48 hours incubation with carnosic acid resulted in hemolysis, an effect reaching statistical significance at 10 µg/ml carnosic acid (Fig. 2).

In order to estimate erythrocyte volume, forward scatter was determined utilizing flow cytometry following a 48 hours incubation in Ringer solution without or with carnosic acid (1- 10 µg/ml). As illustrated in Fig. 3, carnosic acid decreased erythrocyte forward scatter, an effect reaching statistical significance at 10 µg/ml carnosic acid concentration.

In order to estimate cytosolic Ca2+ activity ([Ca2+]i), the erythrocytes were loaded with the Ca2+ sensitive dye Fluo3 and the Fluo3 fluorescence taken as measure of [Ca2+]i. As illustrated in Fig. 4, a 48 hours exposure to carnosic acid increased the Fluo3 fluorescence, an effect reaching statistical significance at 10 µg/ml carnosic acid.

A further series of experiments tested whether the carnosic acid-induced translocation of phosphatidylserine or erythrocyte shrinkage required entry of extracellular Ca2+. To this end, erythrocytes were incubated for 48 hours in the absence or presence of 10 µg/ml carnosic acid in the presence or nominal absence of extracellular Ca2+. As illustrated in Fig. 5, removal of extracellular Ca2+ significantly blunted the effect of carnosic acid on annexin-V-binding. However, carnosic acid significantly increased the percentage of annexin-V-binding erythrocytes even in the absence of extracellular Ca2+. Thus, entry of extracellular Ca2+ accounted for a large part but not for the full effect of carnosic acid on cell membrane scrambling. Removal of extracellular Ca2+ further tended to blunt the effect of carnosic acid on forward scatter, an effect, however not reaching statistical significance. Again, even in the absence of extracellular Ca2+, carnosic acid significantly decreased the erythrocyte forward scatter (Fig. 6). Thus, entry of extracellular Ca2+ presumably contributed to but did not account for the effect of carnosic acid on cell volume.

Stimulators of Ca2+ entry and subsequent eryptosis include oxidative stress. Reactive oxygen species (ROS) was thus quantified utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA). As shown in Fig. 7, a 48 hours exposure to carnosic acid (10 µg/ml) significantly decreased the DCFDA fluorescence. Accordingly, carnosic acid decreased oxidative stress.

Eryptosis could be stimulated without increase of [Ca2+]i by ceramide. Thus, the effect of carnosic acid on ceramide abundance in the cell membrane was estimated by flow cytometry utilizing specific antibodies. As shown in Fig. 8, a 48 hours exposure to carnosic acid (10 µg/ml) significantly increased the ceramide abundance at the erythrocyte surface.

The present observations reveal a novel effect of carnosic acid, i.e. the stimulation of suicidal erythrocyte death or eryptosis. Exposure of human erythrocytes to carnosic acid is followed by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface.

The effect of 10 µg/ml carnosic acid on cell membrane scrambling and cell shrinkage was paralleled by an increase of cytosolic Ca2+ activity ([Ca2+]i), which is known to trigger cell membrane scrambling by activating an illdefined scramblase [58]. An increase of [Ca2+]i is further known to trigger erythrocyte shrinkage by 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 [59]. Comparison of Fig. 1 with Fig. 3 and Fig. 4 reveals that lower concentrations of carnosic acid (2.5 µg/ml) are required to trigger cell membrane scrambling than those required for cell shrinkage and increase of [Ca2+]i. It thus appears that, unlike cell shrinkage, cell membrane scrambling involves a mechanism other than increase of [Ca2+]i.

The effect of high concentrations of carnosic acid was, however, at least in part due to triggering of Ca2+ entry from the extracellular space, as removal of extracellular Ca2+ significantly blunted the effect of carnosic acid on annexin-V-binding. Ca2+ entered presumably through Ca2+ permeable cation channels [58]. The channels may be activated by oxidative stress [58]. However, DCFDA fluorescence reveals that carnosic acid treatment rather decreased the abundance of reactive oxidant species. Thus, the mechanism stimulating the channels remained elusive. The Ca2+ independent triggering of cell membrane scrambling and forward scatter is presumably in part the result of increased abundance of ceramide at the erythrocyte surface.

Besides its effect on eryptosis, carnosic acid also triggered hemolysis. Consequences of eryptosis and hemolysis include the clearance of the defective erythrocytes from circulating blood with potential development of anemia [58]. To the extend that eryptosis precedes hemolysis, eryptosis could prevent or attenuate release of hemoglobin, which otherwise passes the renal glomerular filter, subsequently precipitates in the acidic lumen of renal tubules and thus occludes nephrons [96].

Eryptosis may favourably influence the clinical course of malaria. Following infection with the malaria pathogen Plasmodium, the pathogen imposes oxidative stress on the infected host erythrocyte thus activating Ca2+-permeable erythrocyte cation channels [58,97]. By accelerating eryptosis, sickle-cell trait, beta-thalassemia-trait, Hb-C deficiency and G6PD-deficiency expedite the clearance of infected erythrocytes, thus decreasing parasitemia and protecting against a severe course of malaria [58,98,99,100]. Enhanced eryptosis further decreases parasitemia in iron deficiency [101] and following treatment with lead [101], chlorpromazine [102] or NO synthase inhibitors [102]. It is tempting to speculate that carnosic acid may similarly accelerate eryptosis of Plasmodium infected erythrocytes and thus have the potential to favourably influence the clinical course of the disease.

Besides fostering the development of anemia, eryptosis may lead to adherence of phosphatidylserine exposing erythrocytes to the vascular wall [103], stimulation of blood clotting and triggering of thrombosis [104,105,106]. Eryptosis may thus compromize microcirculation [60,104,107,108,109,110].

Carnosic acid triggers eryptosis with cell shrinkage and cell membrane scrambling, an effect paralleled by and in part due to increase of cytosolic Ca2+ activity and ceramide formation.

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.

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2015
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