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Enhanced programmed cell death of iron-deficient erythrocytes

Department of Physiology, University of Tübingen, Tübingen, Germany

¹ Correspondence: Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, Tübingen 72076, Germany. E-mail: florian.lang{at}uni-tuebingen.de

SPECIFIC AIMS

Iron deficiency is a common disorder leading to the development of anemia in 500 to 600 million people world wide. Iron deficiency-induced anemia results at least in part from reduced erythropoiesis, but may in addition be due to decreased life span of iron-deficient erythrocytes. The present study aimed to elucidate the hitherto poorly understood mechanisms underlying the accelerated clearance of iron-deficient erythrocytes. Specifically, the study analyzed the impact of iron deficiency on eryptosis, a programmed erythrocyte death characterized by erythrocyte shrinkage, membrane blebbing and phosphatidylserine surface exposure, all features typical for apoptotic death of nucleated cells.

PRINCIPAL FINDINGS

1. Iron deficiency increases cation channel activity
The activity of calcium-permeable unselective cation channels has been analyzed in iron-depleted mouse erythrocytes as compared with control mouse erythrocytes using the whole-cell patch-clamp technique. In control erythrocytes as well as in iron-depleted erythrocytes, the spontaneous conductance recorded with K-gluconate pipette solution in combination with NaCl bath solution was very low in both groups. The mean conductances (calculated by linear regression between –100 and 0 mV) were 50 ± 9 pS and 66 ± 11 pS for the control and the iron-depleted mouse erythrocytes, respectively. Replacing the NaCl bath solution by a Cl^–-free solution (Na-gluconate) markedly increased the inward and the outward whole-cell conductances for both groups of mouse erythrocytes without affecting the reversal potentials (Fig. 1 A). Moreover, Cl^– removal induced a more pronounced increase of the inward conductance in the iron-depleted erythrocytes as compared with control erythrocytes. The mean conductance (calculated by linear regression between –100 and 0 mV) reached 172 ± 9 pS in the iron-depleted mouse erythrocytes and only 114 ± 9 pS in the control mouse erythrocytes, a difference that was statistically significant.

View larger version (26K): [in this window] [in a new window]   Figure 1. Iron deficiency-induced cation channel activity, Ca2+ uptake and increase of cytosolic Ca2+ activity in mouse erythrocytes. A) Mean slope conductances ±SE recorded for positive potentials from 0 to +100 mV and for negative potentials between –100 and 0 mV (linear regression). Values were calculated for control mouse erythrocytes (filled bars, n=6) and for iron depleted mouse erythrocytes (open bars, n=5) either in a NaCl (left) or in a Na-gluconate bath solution (right). (t test, *P<0.05). B) Means ±SE (n=4) of time-dependent 45Ca2+ influx (time points: 10 min, 40 min, and 120 min) into erythrocytes from animals receiving control diet (control, filled squares) or from animals receiving an iron deficient diet for 10 wk (low Fe, open squares). [CaT]i values are given as µmol/1013 cells. *Significant difference from the respective control erythrocytes of the same time point (unpaired t test; P0.05). C) Means ±SE (n=4) of Ca2+-dependent fluorescence in FL-1 of erythrocytes from animals receiving control diet (control, filled column) or from animals receiving an iron deficient diet for 10 wk (low Fe, open column) or from ionomycin (1 µM) -treated erythrocytes (Ionomycin, filled column). *Significant difference from the respective control erythrocytes (ANOVA using Dunnett’s test as post hoc test; P0.05).

2. Iron deficiency increases Ca²⁺ entry
Tracer flux experiments revealed a time-dependent uptake of ⁴⁵Ca²⁺, which was increased by 2.4-fold in iron-deficient erythrocytes vs. control erythrocytes (Fig. 1B ). In contrast, the maximum Ca²⁺ loading after treatment of the erythrocytes with 1 µM ionomycin was not altered by the iron-deficient diet.

3. Iron deficiency increases cytosolic Ca²⁺ activity
According to flow cytometric analysis of cells loaded with the Ca²⁺-sensitive Fluo-3 fluorescence dye, erythrocytes from iron-deficient mice showed a shift toward higher fluorescence intensities as compared with control erythrocytes (Fig. 1C ). The mean fluorescence was significantly increased from 7.5 ± 0.2 (n=4) in control cells to 13.0 ± 0.5 (n=4) in iron-deficient erythrocytes. The Ca²⁺ ionophore ionomycin (1 µM) used as a positive control similarly increased Fluo-3 fluorescence intensity of erythrocytes. Thus, cytosolic Ca²⁺ activity was enhanced in erythrocytes from iron-deficient animals.

4. Iron deficiency stimulates phosphatidylserine exposure
Enhanced cytosolic Ca²⁺ activity should lead to phosphatidylserine exposure and increased annexin binding. Indeed, annexin binding of nonstressed erythrocytes (Ringer-treated) was significantly enhanced by 54% (n=24) in erythrocytes from iron-deficient animals as compared with the respective erythrocytes from control animals (Fig. 2 A). Double staining experiments using thiazole orange in combination with Annexin V, Alexa Fluor®568 revealed that the increase of annexin binding was due to enhanced phosphatidylserine exposure of mature erythrocytes and did not result from the observed increase of reticulocytes. In these experiments, the annexin binding of thiazole orange-negative, mature erythrocytes was increased by 56 ± 19% (n=10) in the blood samples from mice, which were fed an iron-depleted diet as compared with the respective control blood samples.

View larger version (26K): [in this window] [in a new window]   Figure 2. Enhanced phosphatidylserine exposure and accelerated clearance of iron-deficient mouse erythrocytes. A) Means ±SE (n=24) of erythrocyte annexin binding after a 12 h exposure to standard Ringer solution. Erythrocytes were from animals receiving control diet (control, filled column) or from animals receiving an iron-deficient diet (low Fe, open column). *Significant difference from the respective erythrocytes from control animals (unpaired t test; P0.05). B) Means ±SE (n=8) of erythrocyte annexin binding after a 12 h exposure to hypertonic fluid (700 mOsm; corresponding to standard Ringer plus 400 mM sucrose). Erythrocytes were from animals receiving control diet (control, filled column) or from animals receiving an iron-deficient diet for 10 wk (low Fe, open column). C) Means ±SE (n=8) of erythrocyte annexin binding after a 12 h exposure to glucose-free solution (0 mM glucose). Erythrocytes were from animals receiving control diet (control, filled column) or from animals receiving an iron-deficient diet for 10 wk (low Fe, open column). *Significant difference from the respective erythrocytes from control animals (unpaired t test; P0.05). D) Means ±SE (n=8) of the percentage of CFSE-labeled control erythrocytes (filled squares) and of iron-depleted erythrocytes (open squares) in peripheral blood after different time points (1, 5, 13, 18, and 25 days) after injection into healthy mice. *Significant difference from the respective control erythrocytes of the same time point (unpaired t test, P0.05).

5. Iron deficiency sensitizes erythrocytes against cell shrinkage and energy depletion
After exposure to hyperosmotic shock, annexin binding was increased in erythrocytes from both control (from 2.4±0.2% to 35.8±1.7%, n=8 each) and iron-deficient animals (from 3.9±0.3%, to 44.2±4.6%, n=8 each). The percentage of annexin binding cells after osmotic shock tended to be higher in iron-deficient erythrocytes as compared with the respective iron replete erythrocytes, a difference, however, not reaching statistical significance (Fig. 2B ). Replacement of extracellular Cl^– with gluconate, which leads to isotonic cell shrinkage and disinhibits the Ca²⁺-permeable cation channel, likewise increased the number of annexin binding erythrocytes from both, control (from 2.4±0.2% to 3.4±0.3%, n=8 each) and iron-deficient animals (from 3.9±0.3% to 8.0±0.9%, n=8 each). The percentage of annexin binding cells after removal of Cl^– was significantly higher in iron-deficient erythrocytes as compared with the respective iron replete erythrocytes. Energy depletion by removal of glucose similarly increased the annexin binding of erythrocytes from both, control (from 2.5±0.1% to 53.4±1.5%, n=8 each) and iron-deficient animals (from 4.0±0.2% to 79.8±2.3%, n=8 each). The percentage of annexin binding cells after glucose depletion was again significantly higher in iron-deficient erythrocytes as compared with the respective iron replete erythrocytes (Fig. 2C ). Taken together, iron-depleted erythrocytes are more susceptible toward cellular stress.

6. Iron deficiency decreases cell volume
Alterations of cell volume of iron replete and iron-depleted erythrocytes were estimated by flow cytometry. As evident from forward scatter analysis, erythrocytes from iron-deficient animals were significantly smaller than erythrocytes from control animals even in the absence of cellular stress. The forward scatter of nonstressed erythrocytes amounted to 544 ± 2 (n=8) in control animals as compared with 392 ± 6 (n=8) in iron-deficient animals. The forward scatter decreased significantly after hyperosmotic shock in both control (to 471±4, n=8) and iron-depleted erythrocytes (to 328 ± 7, n=8). Similarly, glucose depletion decreased forward scatter in control (to 514±3, n=8) and iron-depleted erythrocytes (379±7, n=8). The cell volume of erythrocytes from iron-deficient mice was in all cases (i.e., after hyperosmotic shock, Cl^– removal and energy depletion) significantly smaller than the respective values in erythrocytes from control animals.

7. Ca²⁺ loading and/or iron deficiency decreases in vivo erythrocyte half life
To investigate the impact of enhanced phosphatidylserine exposure on erythrocyte half-life, erythrocytes were labeled with a stable fluorescent dye and fluorescence-labeled cells were injected into healthy mice. Then, peripheral blood was taken after different time points and the percentage of CFSE-positive erythrocytes was determined by flow cytometry. Using this technique, we could demonstrate that annexin-positive cells are rapidly cleared from peripheral blood (not shown). In these experiments, treatment with the Ca²⁺ ionophore ionomycin (1 µM for 1 h) led to annexin binding in 65% of the erythrocytes whereas only 6% of Ringer-treated erythrocytes were annexin-positive. CFSE labeling of Ringer- and ionomycin-treated erythrocytes was very effective with nearly 100% of the cells being CFSE-positive. When those cells were injected into healthy mice 2/3 of the CFSE-labeled, ionomycin-treated cells disappeared from peripheral blood within 2 h after injection whereas the percentage of CFSE-labeled, Ringer-treated erythrocytes remained almost constant. Thus, "apoptotic," annexin-positive erythrocytes are rapidly cleared from peripheral blood. In another set of experiments, the clearance of iron replete, CFSE-labeled and iron- depleted, CFSE-labeled erythrocytes was measured. Clearance of iron-depleted erythrocytes was significantly faster than the clearance of the respective control erythrocytes. As a consequence, the half-life of erythrocytes decreased from 23 days for controls to 15 days for iron-depleted cells (Fig. 2D ).

CONCLUSIONS AND SIGNIFICANCE

Iron deficiency enhances the susceptibility of eythrocytes to undergo programmed cell death or eryptosis. The effect is at least in part due to enhanced activity of the Ca²⁺-permeable cation channel. Ca²⁺ entry through this channel leads to activation of a scramblase with subsequent phosphatidylserine exposure, and to activation of the Gardos channels leading to cellular KCl loss and cell shrinkage. Erythrocytes exposing phosphatidylserine are recognized, bound, engulfed and degraded by macrophages. The present observations provide a mechanistic explanation for the accelerated clearance of iron-deficient erythrocytes from circulating blood. In accordance with the hypothesis that iron deficiency not only affects hematopoiesis but also influences erythrocyte clearance, it is demonstrated here that the half life of CFSE-labeled, iron-deficient erythrocytes is significantly decreased. The enhanced phosphatidylserine exposure of freshly drawn erythrocytes from iron-deficient animals does not only mediate the binding to macrophages but may allow adhesion of erythrocytes to endothelial cells. This adhesion may impede the microcirculation and it is tempting to speculate that it participates in the generation of ischemic complications of iron deficiency.

View larger version (49K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4872fje;

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