Introduction

Pyrogallol, a polyphenolic component of Acacia nilotica [1, 2] has been reported to stimulate apoptosis of cancer cells [3,4,5], fibroblasts [6, 7], endothelial cells [8,9,10], juxtaglomerular cells [11,12,13], U937 cells [14], HEK293 cells [15], K562 cells [15] and blood platelets [2]. Pyrogallol-induced apoptosis involves modification of gene expression [16], nuclear DNA fragmentation [1], mitochondria [1], oxidative stress [2, 3, 7,8,9,10,11,12,13], inhibition of Bcl-2 proteins [17] and caspase activation [2].

Mature, circulating erythrocytes are lacking nuclei and mitochondria but harbor caspases [18, 19] and may enter eryptosis, a suicidal cell death apparent from phosphatidylserine translocation to the outer cell membrane surface [19,20,21,22]. Eryptosis is further typically paralleled by cell shrinkage [23]. Eryptosis may be stimulated by storage [24], energy depletion [19, 25], oxidative stress [19], hyperosmotic shock [19], and excessive Ca2+ entry [19]. Excessive eryptosis may result in anemia, because phosphatidylserine exposing erythrocytes are bound to endothelial cells and removed from circulating blood [26, 27]. Eryptosis and subsequent removal of Plasmodium-infected erythrocytes may, on the other hand, reduce parasitemia and thus be beneficial in malaria [28]. Accordingly, small molecules stimulating [29,30,31,32,33,34] and small molecules inhibiting [25, 29, 30, 35] eryptosis may be of clinical interest.

The aim of the present analysis was to disclose a potential stimulating or inhibiting effect of pyrogallol on eryptosis. Human erythrocytes isolated from healthy volunteers were incubated in normal Ringer or in absence of glucose (energy depletion), in presence of tert-butyl-hydroperoxide (t-BOOH, oxidative stress), in hyperosmotic Ringer (sucrose added) or in presence of Ca2+ ionophore ionomycin, each without or with addition of pyrogallol. After treatment, flow cytometry was employed to determine abundance of phosphatidylserine at the erythrocyte surface and erythrocyte volume.

Materials and methods

Fresh Li-Heparin-anticoagulated blood drawn from healthy volunteers was kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). The blood was centrifuged at 120 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, 5 glucose, 1 CaCl2, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acids (HEPES; pH 7.4) at 37 °C for 48 h. Where indicated, erythrocytes were exposed for 48 h to glucose containing or glucose depleted Ringer solution, for 30 min to the oxidant t-BOOH (0.5 mM, Sigma Aldrich, Hamburg, Germany), for 6 h to hypertonic Ringer (addition of 550 mM sucrose, Sigma Aldrich, Hamburg, Germany) or for 60 min to Ca2+ ionophore ionomycin (1 µM, Merck Millipore, Darmstadt, Germany), each in the absence and presence of pyrogallol (2–8 µM, Sigma Aldrich, Hamburg, Germany). Exposure time to glucose depletion as well as concentrations of and exposure times to t-BOOH, sucrose and ionomycin have been chosen according to previous experiences [36], concentrations of pyrogallol according to studies on the effect of this substance in other cell types [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]

After incubation under the respective experimental condition, a 150 Annexin-V-FITC 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 20 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 pyrogallol treated erythrocytes [36].

For each parameter, 50,000 events were counted. The analysis of FACS data was performed using FlowJo software v10.0.7 (FlowJo, Ashland, USA). 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 or 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 [36].

Results

Following incubation for 48 h in standard Ringer solution, the percentage of annexin-V-binding erythrocytes was similarly low without (1.06 ± 0.07%, n = 13) and with (1.19 ± 0.10%, n = 13) presence of pyrogallol (8 µM). The forward scatter was again similar following a 48 h exposure to standard Ringer solution without (480.96 ± 4.73, n = 13) or with (476.86 ± 4.28%, n = 13) pyrogallol (8 µM). Thus, in standard Ringer solution, pyrogallol did not significantly alter phosphatidylserine abundance at the erythrocyte surface or erythrocyte volume.

Energy depletion by a 48 h exposure to Ringer without glucose was followed by a significant increase of the percentage of annexin-V-binding erythrocytes (Fig. 1a). Addition of pyrogallol (2–8 µM) to glucose-free Ringer significantly blunted, but did not abrogate the increase of the percentage of annexin-V-binding erythrocytes (Fig. 1b). Thus, pyrogallol blunted, but did not prevent phosphatidylserine translocation following energy depletion.

Fig. 1
figure 1

Pyrogallol sensitivity of phosphatidylserine exposure and cell volume following energy depletion. a Original histograms of annexin-V-binding of erythrocytes following exposure for 48 h to glucose containing Ringer solution (red line), Ringer solution without glucose (blue line) and Ringer solution without glucose in the presence of pyrogallol (8 µM) (purple line); b Arithmetic means ± SEM (n = 13) of the percentage annexin-V-binding erythrocytes after a 48 h treatment with Ringer solution with (left bar, Control) or without (right bars) glucose in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates effect of solvent; c Original histograms of erythrocyte forward scatter following exposure for 48 h to glucose containing Ringer solution (red line), Ringer solution without glucose (blue line) and Ringer solution without glucose in the presence of pyrogallol (8 µM) (purple line); d Arithmetic means ± SEM (n = 13) of the erythrocyte forward scatter after a 48 h treatment with Ringer solution with (left bar, Control) or without (right bars) glucose in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates the effect of solvent ***(p < 0.001) indicates significant difference from the presence of glucose, ###(p < 0.001) indicates significant difference from the absence of pyrogallol (ANOVA). (Color figure online)

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Energy depletion by a 48 h exposure to Ringer without glucose was further followed by a significant decrease of forward scatter, which was virtually identical in the absence and presence of pyrogallol (2–8 µM) (Fig. 1c). Thus, pyrogallol did not appreciably alter the shrinking effect of energy depletion (Fig. 1d).

Oxidative stress was induced by treatment with t-BOOH. Within 30 min 0.5 mM t-BOOH triggered a sharp significant increase of the percentage of annexin-V-binding erythrocytes (Fig. 2a). The effect was slightly, but significantly, blunted in the presence of pyrogallol (2–8 µM). However, in the presence of pyrogallol, t-BOOH still significantly increased the percentage of phosphatidylserine exposing erythrocytes (Fig. 2b). Thus, pyrogallol blunted, but did not prevent phosphatidylserine translocation following oxidative stress.

Fig. 2
figure 2

Pyrogallol sensitivity of phosphatidylserine exposure and cell volume following oxidative stress. a Original histograms of annexin-V-binding of erythrocytes following exposure for 30 min to Ringer solution without (red line) or with 0.5 mM t-BOOH without (blue line) and with purple line) presence of pyrogallol (8 µM); b Arithmetic means ± SEM (n = 8) of the percentage annexin-V-binding erythrocytes after a 30 min treatment with Ringer solution without (left bar, Control) or with 0.5 mM t-BOOH (right bars) in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates effect of solvent. c Original histograms of erythrocyte forward scatter following exposure for 30 min to Ringer solution without (red line) or with 0.5 mM t-BOOH without (blue line) and with purple line) presence of pyrogallol (8 µM); d Arithmetic means ± SEM (n = 8) of the erythrocyte forward scatter after a 48 h treatment with Ringer solution without 0.5 mM t-BOOH (left bar, Control) or with 0.5 mM t-BOOH (right bars) in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates effect of solvent. ***(p < 0.001) indicates significant difference from the absence of t-BOOH, #(p < 0.05), ##(p < 0.01) indicates significant difference from the absence of pyrogallol (ANOVA). (Color figure online)

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Within 30 min, 0.5 mM t-BOOH caused a slight, but significant, decrease of forward scatter (Fig. 2c). The presence of pyrogallol did not significantly modify the decrease of forward scatter following t-BOOH treatment and in the presence of pyrogallol, t-BOOH still significantly decreased the forward scatter of erythrocytes (Fig. 2d). Thus, pyrogallol did not significantly modify the shrinking effect of oxidative stress.

For induction of hyperosmotic shock, Ringer was made hypertonic by the addition of 550 mM sucrose. A 6 h treatment with hypertonic Ringer solution was followed by a significant increase of the percentage of annexin-V-binding erythrocytes (Fig. 3a). The effect was blunted, but not abrogated, in the presence of pyrogallol (2–8 µM) (Fig. 3b). Thus, pyrogallol blunted, but did not prevent phosphatidylserine translocation following hyperosmotic shock.

Fig. 3
figure 3

Pyrogallol sensitivity of phosphatidylserine exposure and cell volume following hyperosmotic shock. a Original histograms of annexin-V-binding of erythrocytes following exposure for 6 h to Ringer solution without (red line) or with added 550 mM sucrose without (blue line) and with purple line) presence of pyrogallol (8 µM); b Arithmetic means ± SEM (n = 8) of the percentage annexin-V-binding erythrocytes for 6 h treatment with Ringer solution without (left bar, Control) or with added 550 mM sucrose (right bars) in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates effect of solvent; c Original histograms of erythrocyte forward scatter following exposure for 6 h to Ringer solution without (red line) or with added 550 mM sucrose without (blue line) and with purple line) presence of pyrogallol (8 µM); d Arithmetic means ± SEM (n = 8) of the erythrocyte forward scatter after a for 6 h treatment with Ringer solution without added 550 mM sucrose (left bar, Control) or with 550 mM sucrose (right bars) in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates the effect of solvent. ***(p < 0.001) indicates significant difference from isotonic Ringer, #(p < 0.05), ##(p < 0.01), ###(p < 0.001) indicates significant difference from the absence of pyrogallol (ANOVA). (Color figure online)

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A 6 h treatment with hypertonic Ringer induced a sharp significant decrease of forward scatter (Fig. 3c). The presence of pyrogallol did not significantly modify the decrease of forward scatter following treatment with hypertonic Ringer and in the presence of 8 µM pyrogallol, hypertonic Ringer still significantly decreased the forward scatter of the erythrocytes (Fig. 3d). Thus, pyrogallol did not significantly modify the shrinking effect of hyperosmotic shock.

Ca2+ overload was accomplished by treatment with Ca2+ ionophore ionomycin. Within 60 min 1 µM ionomycin triggered a sharp significant increase of the percentage of annexin-V-binding erythrocytes (Fig. 4a). The effect was significantly blunted, but not abolished in the presence of pyrogallol (2–8 µM) (Fig. 4-B). Thus, pyrogallol blunted, but did not prevent phosphatidylserine translocation following Ca2+ overload.

Fig. 4
figure 4

Pyrogallol sensitivity of phosphatidylserine exposure and cell volume following Ca2+ overload. a Original histograms of annexin-V-binding of erythrocytes following exposure for 60 min to Ringer solution without (red line) or with 1 µM ionomycin without (blue line) and with purple line) presence of pyrogallol (8 µM); b Arithmetic means ± SEM (n = 7) of the percentage annexin-V-binding erythrocytes after a 48 h treatment with Ringer solution without (left bar, control) or with 1 µM ionomycin (right bars) in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates effect of solvent; c Original histograms of erythrocyte forward scatter following exposure for 60 min to Ringer solution without (red line) or with 1 µM ionomycin without (blue line) and with purple line) presence of pyrogallol (8 µM); d Arithmetic means ± SEM (n = 7) of the erythrocyte forward scatter after a 48 h treatment with Ringer solution without (left bar, Control) or with 1 µM ionomycin (right bars) in the absence (blue bar) and presence (purple bars) of pyrogallol (2–8 µM). DMSO (black bar) indicates effect of solvent. ***(p < 0.001) indicates significant difference from the absence of ionomycin, ###(p < 0.001) indicates significant difference from the absence of pyrogallol (ANOVA). (Color figure online)

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Within 60 min 1 µM ionomycin induced a sharp, significant decrease of forward scatter (Fig. 4c). The presence of pyrogallol did not significantly modify the decrease of forward scatter following ionomycin treatment and in the presence of 8 µM pyrogallol, ionomycin still significantly enhanced the percentage of phosphatidylserine exposing erythrocytes (Fig. 4d). Thus, pyrogallol did not significantly modify the shrinking effect of Ca2+ overload.

Discussion

The present observations reveal that pyrogallol could inhibit phosphatidylserine translocation to the erythrocyte surface, a hallmark of eryptosis [19]. Without maneuvers stimulating eryptosis pyrogallol did not affect phosphatidylserine translocation. The substance blunted, however, significantly the phosphatidylserine translocation following glucose deprivation, oxidation, hyperosmotic shock, and Ca2+ loading. Those maneuvers have been shown in a variety of previous studies to trigger eryptosis [19].

Accelerated eryptosis causes removal of circulating erythrocytes and anemia in diverse clinical conditions including chronic kidney disease [37], HUS (hemolytic uremic syndrome) [38], Autoimmune Haemolytic Anaemia [39], iron deficiency [19], hyperphosphatemia [40], vitamin D excess [41], dehydration [42], diabetes [43], hepatic failure [44], malignancy [45], sepsis [46], sickle cell anemia [19], beta-thalassemia [19], Hb-C and G6PD-deficiency [19], as well as Wilson´s disease [46]. The clearance of eryptotic erythrocytes [19] results in anemia as soon as it surpasses the rate of erythropoiesis [19]. Excessive eryptosis is further expected to compromise microcirculation [47], because eryptotic erythrocytes adhere to endothelial cells [48], and favour development of thrombosis [49]. Inhibitors of eryptosis are expected to reverse anemia and improve microcirculation in disorders with accelerated eryptosis. On the other hand, interference with eryptosis may delay removal of defective circulating erythrocytes which may instead enter hemolysis [19]. Hemoglobin thus released may undergo glomerular filtration with subsequent tubular hemoglobin precipitation, nephron occlusion, and thus damage of the kidneys [50].

Conclusions

Pyrogallol blunts the stimulation of eryptotic phosphatidylserine translocation to the erythrocyte surface following energy depletion, oxidative stress, hyperosmotic cell shrinkage, and Ca2+ overload.