The pathogen of malaria, Plasmodium, enters erythrocytes and thus escapes recognition by the immune system. The pathogen induces oxidative stress to the host erythrocyte, which triggers eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are identified by macrophages which engulf and degrade the eryptotic cells. To the extent that infected erythrocytes undergo eryptosis prior to exit of Plasmodiaand subsequent infection of other erythrocytes, the premature eryptosis may protect against malaria. Accordingly, any therapeutical intervention accelerating suicidal death of infected erythrocytes has the potential to foster elimination of infected erythrocytes, delay the development of parasitemia and favorably influence the course of malaria. Eryptosis is stimulated by a wide variety of triggers including osmotic shock, oxidative stress, energy depletion and a wide variety of xenobiotics. Diseases associated with accelerated eryptosis include sepsis, haemolytic uremic syndrome, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency and Wilson’s disease. Among the known stimulators of eryptosis, paclitaxel, chlorpromazine, cyclosporine, curcumin, PGE₂ and lead have indeed been shown to favourably influence the course of malaria. Moreover, sickle-cell trait, beta-thalassemia trait, glucose-6-phosphate dehydrogenase (G6PD)-deficiency and iron deficiency confer some protection against a severe course of malaria. Importantly, counteracting Plasmodia by inducing eryptosis is not expected to generate resistance of the pathogen, as the proteins involved in suicidal death of the host cell are not encoded by the pathogen and thus cannot be modified by mutations of its genes.

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apcMin/+) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca2+ activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin–luciferase in isolated erythrocytes from apcMin/+ mice and wild-type mice (apc+/+). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apcMin/+ mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Extracellular Ca2+ removal or inhibition of Ca2+ entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca2+-entry by treatment with Ca2+-ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Following retrieval and injection into the circulation of the same mice, apcMin/+ erythrocytes were more rapidly cleared from circulating blood than apc+/+ erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apcMin/+ mice. The observations point to accelerated eryptosis and subsequent clearance of apcMin/+ erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.

Janus kinase 3, a tyrosine kinase expressed in haematopoetic tissues, plays a decisive role in Tlymphocyte survival. JAK3 deficiency leads to (Severe) Combined Immunodeficiency (SCID) resulting from enhanced lymphocyte apoptosis. JAK3 is activated by phosphorylation. Nothing is known about expression of JAK3 in erythrocytes, which may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure and cell shrinkage. Triggers of eryptosis include energy depletion. The present study utilized immunohistochemistry and confocal microscopy to test for JAK3 expression and phosphorylation, and FACS analysis to determine phosphatidylserine exposure (annexin binding) and cell volume (forward scatter). As a result, JAK3 was expressed in erythrocytes and phosphorylated following 24h and 48h glucose depletion. Forward scatter was slightly but significantly smaller in erythrocytes from JAK3-deficient mice (jak3 -/-) than in erythrocytes from wild type mice (jak3). Annexin V binding was similarly low in both genotypes. The JAK3 inhibitors WHI-P131/JANEX-1 (4-(4'-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 156μM) and WHI-P154 (4-[(3'-Bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 11.2μM) did not significantly modify annexin V binding or forward scatter. Glucose depletion increased annexin V binding, an effect significantly blunted in jak3 -/-erythrocytes and in the presence of the JAK3 inhibitors. The observations disclose a completely novel role of Janus kinase 3, i.e. the triggering of cell membrane scrambling in energy depleted erythrocytes.

The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFĸB, by inhibiting NFĸB directly (parthenolide) or by interfering with the inactivation of the NFĸB inhibitory protein IĸB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca²⁺ leakage or glutathione depletion. The present study utilized Western blotting to search for NFĸB and IĸB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca²⁺ (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFĸB and IĸB-α are expressed in erythrocytes. Targeting the NFĸB pathway by Bay 11-7082 (IC₅₀ ≈ 10 µM) and parthenolide (IC₅₀ ≈ 30 µM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca²⁺ and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFĸB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFĸB-dependent gene expression.

Α-lipoic acid, a nutrient with both, antioxidant and oxidant activity induces apoptosis in a variety of cells. Owing to its proapoptotic potency Α-lipoic acid has been suggested for the therapy of cancer. Α-Lipoic acid stimulates apoptosis by induction of oxidative stress and subsequent activation of caspases. Oxidative stress could similarly trigger caspase activation and suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling and cell shrinkage. Eryptosis is triggered by increase of cytosolic Ca²⁺ concentration and/or ceramide formation. The present study explored whether Α -lipoic acid influences eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in FACS analysis, cytosolic Ca²⁺ concentration from Fluo3 fluorescence, caspase activation and ceramide formation utilizing respective antibodies, cytosolic ATP concentration from a luciferase-assay. Within 48 hours, exposure to Α-lipoic acid (10 - 75 mM) significantly decreased forward scatter, increased cytosolic Ca²⁺ concentration, decreased ATP concentration, activated caspase 3, stimulated formation of ceramide and triggered annexin V-binding. Glucose depletion (48 h) was followed by decrease of forward scatter and increase of annexin V-binding, effects significantly augmented in the presence of Α-lipoic acid (20 mM). Oxidative stress (30 min 0.3 mM tert-butylhydroperoxide) similarly triggered annexin binding, an effect slightly but significantly blunted by Α-lipoic acid. In conclusion, Α-lipoic acid triggers eryptosis but by the same token counteracts eryptosis during oxidative stress. Α-lipoic acid sensitive eryptosis may lead to anemia and derangements of microcirculation.