Enhanced Eryptosis Following Exposure to Lopinavir

The protease inhibitor lopinavir is used for the treatment of HIV infections [1,2,3,4,5,6,7,8,9,10,11,12,13]. Side effects of lopinavir treatment include anemia [14]. Lopinavir may induce proteotoxic stress [15,16], trigger oxidative stress [16], and suppress NF-κB activity [17]. Lopinavir may thus foster apoptosis and inhibit growth of tumor cells [15,16,17,18].

Oxidative stress may similarly trigger eryptosis, the suicidal death of erythrocytes, characterized by cell shrinkage [19] and phospholipid scrambling of the cell membrane, which is apparent from phosphatidylserine translocation to the cell surface [20]. Oxidative stress is in part effective by opening of Ca²⁺ permeable unselective cation channels with subsequent Ca²⁺ entry and increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) [20]. Eryptosis is further triggered by ceramide [21], energy depletion [20], and caspases [20,22,23]. Eryptosis is stimulated by several kinases including casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, and p38 kinase [20]. Eryptosis is inhibited by AMP activated kinase AMPK, cGMP-dependent protein kinase, PAK2 kinase, and sorafenib/sunitinib sensitive kinases [20]. Triggers of eryptosis further include a wide variety of xenobiotics [20,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49]. Enhanced eryptosis is observed in several clinical conditions, such as chronic kidney disease (CKD) [29,50,51,52], hemolytic-uremic syndrome [53], sepsis [54], dehydration [37], hyperphosphatemia [47], hepatic failure [55], diabetes [56], malignancy [20], malaria [20,57,58,59], sickle-cell disease [20], beta-thalassemia [20], Hb-C and G6PD-deficiency [20], as well as Wilsons disease [60].

The present study explored whether lopinavir triggers eryptosis. To this end, human erythrocytes from healthy volunteers were exposed for 48 hours to lopinavir and phosphatidylserine surface abundance, cell volume, [Ca²⁺]_i, reactive oxygen species, GSH abundance 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 for 48 hours 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. Where indicated, erythrocytes were exposed to lopinavir (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 in FL-1 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 lopinavir 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”.

In order to determine hemolysis, the samples were centrifuged (3 min at 1600 rpm, room temperature) after incubation under the respective experimental conditions and the supernatants were harvested. As a measure of hemolysis, the haemoglobin (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.

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. Then, Ca²⁺-dependent fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.

Oxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 150 µl suspension of erythrocytes was washed in Ringer solution and then stained with DCFDA (Sigma Aldrich, Hamburg, 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 two times in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 µl Ringer solution, and ROS-dependent fluorescence intensity was measured in FL-1 at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).

The content of reduced glutathione was measured using mercury orange. After incubation, erythrocytes were washed in Ringer solution and then loaded with 40 µM mercury orange (Sigma-Aldrich, Hamburg, Germany) in PBS and incubated at 37°C for 3 mins. Afterwards, the samples were washed once and were finally resuspended in 200 µl PBS. The fluorescence intensity was measured in FL-2 channel by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 576 nm.

To determine the ceramide abundance at the erythrocyte surface, a monoclonal antibody was used. After incubation, cells were stained for 1 h at 37°C with 1μg/ml anti-ceramide antibody (clone MID 15B4; Alexis, Grünberg, Germany) in phosphate-buffered saline (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 in FL-1 at 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 tested whether lopinavir stimulates suicidal erythrocyte death or eryptosis. The two hallmarks of eryptosis are cell shrinkage and phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the cell surface. Annexin-V-binding was determined by flow cytometry as a measure of phosphatidylserine exposure at the erythrocyte surface. The measurements were made following incubation for 48 hours in Ringer solution without or with lopinavir (5 - 20 µg/ml). As illustrated in Fig. 1, a 48 hours exposure to lopinavir was followed by an increase of phosphatidylserine exposing erythrocytes, an effect reaching statistical significance at 10 µg/ml lopinavir.

In order to identify shrunken erythrocytes, erythrocyte volume was estimated from forward scatter, which was determined utilizing flow cytometry. Again, the measurements were made following a 48 hours incubation in Ringer solution without or with lopinavir (5 - 20 µg/ml). As apparent from Fig. 2, lopinavir treatment was followed by a decrease of erythrocyte forward scatter, an effect reaching statistical significance at 15 µg/ml lopinavir concentration.

In order to estimate the effect of lopinavir on hemolysis, the haemoglobin concentration in the supernatant was determined. As a result, following a 48 hours incubation the percentage hemolysed erythrocytes inceased from 1.9 ± 0.2 % (n = 4) in the absence of lopinavir to 3.6 ± 0.4 % (5 µg/ml), 7.9 ± 1.8 % (10 µg/ml), 15.7 ± 2.2 % (15 µg/ml) and 28.8 ± 6.7 % (20 µg/ml) in the presence of lopinavir (n = 4 each). The effect reached statistical significance at 15 µg/ml lopinavir concentration.

Cytosolic Ca²⁺ activity ([Ca²⁺]_i) was estimated utilizing Fluo3 fluorescence. As illustrated in Fig. 3, a 48 hours exposure to lopinavir was followed by an increase of the Fluo3 fluorescence, an effect reaching statistical significance at 20 µg/ml lopinavir concentration.

In order to test whether the effect of lopinavir on phosphatidylserine translocation required entry of extracellular Ca²⁺, erythrocytes were incubated for 48 hours in the absence or presence of 20 µg/ml lopinavir in the presence or nominal absence of extracellular Ca²⁺. As apparent from Fig. 4, removal of extracellular Ca²⁺ significantly blunted the effect of lopinavir on annexin-V-binding. However, even in the absence of extracellular Ca²⁺, lopinavir significantly increased the percentage of annexin-V-binding erythrocytes. Thus, lopinavir triggered erythrocyte cell membrane scrambling in part but not exclusively by stimulating entry of extracellular Ca²⁺.

Stimulators of Ca²⁺ entry and eryptosis include oxidative stress. DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) was thus employed to quantify reactive oxygen species (ROS). As illustrated in Fig. 5, a 48 hours exposure to lopinavir (20 µg/ml) was followed by a significant increase of DCFDA fluorescence. Accordingly, lopinavir induced oxidative stress.

The enhanced oxidative stress could have resulted from decreased GSH levels leading to an impaired anti-oxidative stress defense. Thus, mercury orange staining was employed to quantify GSH abundance. As illustrated in Fig. 6, the mercury orange-dependent fluorescence was significantly lower after exposure to 20 µg/ml lopinavir for 48 hours compared to exposure to Ringer solution.

Additional experiments addressed the impact of lopinavir on ceramide abundance at the erythrocyte surface. The ceramide abundance was quantified utilizing specific antibodies. As a result, following a 48 hours incubation, the ceramide abundance was similar following incubation with 20 µg/ml lopinavir (15.6 ± 0.5 a.u., n = 11) and in the absence of lopinavir (15.7 ± 0.4 a.u., n = 11).

The present observations unravel a novel effect of lopinavir, i.e. the stimulation of eryptosis, the suicidal erythrocyte death. Exposure of human erythrocytes drawn from healthy individuals to lopinavir is followed by cell shrinkage and phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. The effect of lopinavir on eryptosis was accompanied by enhanced hemolysis. The concentrations required for the effect are well in the range of plasma concentrations determined in patients under lopinavir treatment [61]. The observed eryptosis and hemolysis may thus well explain the anemia following lopinavir treatment [14].

The effect of lopinavir on cell membrane scrambling was obviously in large part due to increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) resulting from Ca²⁺ entry from the extracellular space. Accordingly, removal of extracellular Ca²⁺ significantly blunted the lopinavir-induced cell membrane scrambling. Ca²⁺ entered presumably through Ca²⁺ permeable cation channels, which are known to be activated by oxidative stress [20]. As apparent from the increase of DCFDA fluorescence, lopinavir treatment did increase the abundance of reactive oxygen species. The increase in oxidative stress was paralleled by a decreased GSH abundance.

The increase of [Ca²⁺]_i presumably further accounts for the observed lopinavir-induced erythrocyte shrinkage, as an increase of [Ca²⁺]_i leads 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 [19].

The stimulation of eryptosis by lopinavir may lead to anemia, as long as the formation of new erythrocytes cannot match the loss of eryptotic erythrocytes [20]. Moreover, phosphatidylserine exposing erythrocytes adhere to the vascular wall [62], stimulate blood clotting, and trigger thrombosis [63,64,65]. The stimulation of eryptosis by lopinavir may thus compromize microcirculation [21,63,66,67,68,69].

In conclusion, lopinavir triggers eryptosis with cell shrinkage and cell membrane scrambling, an effect paralleled by and in part due to induction of oxidative stress and increase of cytosolic Ca²⁺ activity.

The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch. The study was supported by the Deutsche Forschungsgemeinschaft.

The authors of this manuscript state that they do not have any conflict of interests and nothing to disclose.

R. Bissinger and S. Waibel contributed equally and thus share first authorship.