Normal Mouse Red Blood Cell Research Articles

Yippee like 4 (Ypel4) is essential for normal mouse red blood cell membrane integrity

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

Sickle Cell Disease Is Associated with Reduced Adenosine Deaminase Catalytic Activity, Resulting in Altered Adenosine Metabolism

Abstract 1078Sickle cell disease (SCD), a chronic hemolytic anemia due to a mutant beta-globin, is associated with prominent inflammatory features with elevated levels of inflammation mediating molecules and an oxidative state, with elevated levels of reactive oxygen species (ROS). Additionally, elevated levels of adenosine (Ado) have recently been shown to play a role in exacerbating sickle cell pathophysiology. However, the etiology of increased Ado is unclear; and presumed to occur secondary to increased ATP release from cellular damage (Xu et al, Nature Medicine, 2010). Herein, we show that increased levels of Ado in the plasma of patients with SCD and the mouse model of SCD [Berkeley sickle (SS) mice] were accompanied by significantly reduced catalytic activity of the Ado degrading enzyme, adenosine deaminase (ADA) in sickle red blood cell (RBCs). ADA activity in Berkeley sickle (SS) RBCs was 246 ± 50 U/L as compared to 570 ± 36 U/L in normal mouse RBCs (derived from Berkeley AA/WT mice), as measured by a color-based enzymatic assay. Notably, despite lower enzyme activity of ADA, immunoblotting assays showed that the levels of ADA protein in sickle RBC were similar, or even slightly higher compared to that in wild type (WT) RBC, suggesting that ADA catalytic activity was impaired in sickle RBCs. In vitro plasma swap experiments, where WT RBC are incubated with sickle plasma and vice versa for an hour, revealed that sickle plasma induced a significant decrease in ADA activity in WT RBC, suggesting that plasma from sickle mice contains a soluble factor that is capable of impairing ADA enzyme activity. Interestingly enough, the decrease in ADA activity correlated inversely with the levels of intracellular RBC ROS in sickle mice. Next we directly assess in vivo the effect of induced oxidative stress on RBC ADA activity in sickle mice by injecting sickle mice with 35umoles/Kg body weight of hemin and saline respectively. Preliminary data suggests a decrease in RBC ADA activity in hemin–injected mice (294 ± 197 U/L) compared to the saline-injected group (346 ± 185.8 U/L), and this data was consistent with a concomitant increase in intracellular RBC and white blood cells ROS levels. We conclude that increase in oxidative stress levels mediates a post translational alteration of ADA catalytic activity and alters Ado metabolism in SCD. Disclosures:No relevant conflicts of interest to declare.

Enhanced Phosphatidylserine Scrambling in Oxidatively Challenged Red Blood Cells.

Red blood cells (RBC) that abnormally expose phosphatidylserine (PS) contribute to the pathophysiology of several hemoglobinopathies. PS exposure requires inactivation of the flippase that transports PS from the outer to the inner membrane monolayer, and activation of a phospholipid scrambling process. To evaluate the role of increased oxidative stress in this process, we compared RBC from transgenic sickle mice (Berkeley type) with RBC from peroxiredoxin 2 (prdx) knock-out mice (prdx −/−). These mice lack one of the most prevalent cytosolic antioxidant molecules. This molecule, also known as calpromotin, was previously implicated in membrane abnormalities of the dense sickle RBC population. Mice that lack prdx are slightly anemic, have reduced RBC survival and exhibit a subpopulation of older highly oxidized RBC. The prdx −/− strain did not show a subpopulation of PS-exposing cells in freshly collected blood and the flippase activity, measured by transbilayer kinetics of the fluorescent probe NBD-PS, was normal. In contrast, blood collected from the sickle mice showed a large subpopulation with decreased flippase activity and exhibited a subpopulation of PS-exposing RBC that lack flippase activity. Flippase inhibition induced with vanadate or NEM did not increase the PS exposure of prdx −/− RBC incubated with high levels of Ca2+, indicating that there was no increased Ca2+ influx. In sickle cells, elevated intracellular Ca2+ was evident under similar loading conditions. Loading RBC with 0.1 mM Ca2+, but not lower concentrations, using Ca-ionophore resulted in bilayer scrambling and PS exposure in both strains as well as in normal control mice. The rate of PS scrambling was increased 1.5-fold in sickle mice compared to normal mouse RBC. While the scrambling rate was normal in the young, not oxidized prdx−/− RBC, it was increased 3-fold in the older highly oxidized prdx −/− RBC as compared to normal mouse RBC. The sulfhydryl modifiers NEM or PDA caused flippase inhibition, and altered the PS scrambling rate in normal mouse RBC as reported earlier. Both sickle cells and the older oxidized prdx −/− RBC showed a reduced susceptibility to NEM and PDA, while the younger prdx −/− RBC exhibited a normal sensitivity to these compounds. This suggests that both prdx −/− RBC and sickle cells have sustained similar sulfhydryl damage leading to enhanced scrambling. Exposure to three well-known oxidants (0.1–0.5 mM cumene hydroperoxide, tert-butyl hydroperoxide or hydrogen peroxide) did not increase the percentage of oxidized cells or PS exposure in prdx −/− RBC compared to normal RBC. This indicates that targeted sulfhydryl modification but not general short-term oxidative stress impacts the loss of phospholipid asymmetry. These data confirm that increased oxidative sulfhydryl damage results in a higher propensity for phospholipid scrambling. The presence of active prdx is important to maintain PS asymmetry as it prevents accelerated phospholipid scrambling. In those cells in which the flippase is also inactivated, PS exposure becomes apparent. The loss of flippase activity is much more prevalent in sickle RBC, indicating that prdx does not play an important role in protecting the flippase from inactivation. It can be expected that PS-exposing cells are rapidly removed from the circulation, as they resemble apoptotic cells, which may explain their absence in blood from prdx−/− mice. The presence of PS-exposing RBC in the circulation of sickle mice suggests that the formation of these cells overwhelms their removal.

Targeted Deletion of α-Adducin Results in Absent β-Adducin, Compensated Hemolytic Anemia, and Hydrocephalus in Mice.

The membrane skeleton underlies the lipid bilayer and imparts strength and deformability to the red blood cell (RBC). Spectrin, the major component of the membrane skeleton, is crosslinked by short actin filaments into a two-dimensional array at junctional complexes, which are composed of multiple additional proteins including adducin. Three mammalian adducins (α, β, γ) exist that are encoded by distinct genes (Add1, Add2, Add3). α- and γ-adducin are ubiquitously expressed, with the highest expression of α-adducin seen in erythroid cells, brain, kidney, and heart. Expression of β-adducin is restricted to erythroid cells and brain. In RBCs heterotetramers of α- and β-adducin regulate actin filament length via barbed end capping. Previously, we deleted β-adducin in mice. Loss of β-adducin resulted in decreased α-adducin and up-regulated γ-adducin (5-fold), producing fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Here, we report on recently generated α-adducin null mice. We inactivated the α-adducin gene (Add1) by targeted deletion of exons 10–12, and confirmed by western blotting the complete loss of α-adducin in RBC ghosts and in the brain. All mice were maintained on a segregating B6.129 genetic background. Heterozygous mice are normal in all parameters examined to date. Notably, although β-adducin gene expression is normal, western blotting revealed a complete absence of β-adducin protein in α-adducin null RBC ghosts. γ-adducin, present at low levels in normal mouse RBCs, was unchanged, as were all other membrane skeleton proteins examined by SDS-PAGE and/or western blotting (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). α-adducin null mice display characteristics of mild compensated hemolytic anemia. The hematocrit is significantly decreased (43 vs. 46% in wildtype). RBCs are microcytic (MCV 41.9 vs. 46.4 fL) and osmotically fragile (50% lysis at 215 mOsm NaCl vs. 190 mOsm). The MCH is significantly decreased while the MCHC is significantly elevated, suggestive of RBC dehydration. The percentage of circulating reticulocytes is significantly increased (5.0 vs. 2.7%). Spleen weights are normal. Examination of peripheral blood smears and scanning electron microscopy confirms microcytic, anisocytotic RBCs with spherocytes and elliptocytes present. α-adducin null mice show postnatal growth retardation and approximately 66% develop critical hydrocephalus with marked expansion of the lateral and third ventricles by the fourth month of age. Normal littermates never show hydrocephalus. We conclude:1.α-adducin null mice have mild, compensated hemolytic anemia;2.β-adducin is unstable in RBCs in the absence of α-adducin;3.loss of α-adducin in the brain is associated with a high incidence of lethal hydrocephalus.Additional studies of the α-adducin null mouse model will be useful in defining protein functions and interactions in RBCs, the requirement for adducin in platelet function, the role of adducin in the brain, and its role in the regulation of systemic blood pressure.

Targeted Deletion of γ-Adducin in Mice.

The adducins are a family of three closely related proteins (α, β, γ) encoded by distinct genes. α- and γ-adducin are expressed ubiquitously, while β expression is restricted to hematopoietic cells and the brain. In red blood cells (RBCs) adducin localizes to spectrin-actin junctions in the membrane skeleton as αβ heterotetramers. Previously (Gilligan et. al., PNAS, 1999) we showed that deletion of β-adducin results in osmotically fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced in β-adducin null RBCs. We also demonstrated that γ-adducin is present in low amounts in normal mouse RBCs and is upregulated ∼5-fold in β-adducin null RBCs. The increase in γ-adducin suggests that αγ heterotetramers may be compensating for the absence of β-adducin. In an effort to analyze γ-adducin function in RBCs in greater detail, we generated a conditional γ-adducin knockout allele in mice using a Cre-loxP strategy to delete exon 2 containing the start codon. All mice were maintained on a segregating B6.129 genetic background. Western blotting confirmed the absence of γ-adducin in spleen homogenates and RBC ghost preparations from γ-adducin null mice. All other membrane skeleton proteins examined by a combination of SDS-PAGE and western blotting, including α- and β-adducin, are normal in γ-adducin null RBCs (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). Phenotypically, γ-adducin null mice display normal growth curves and show no overt defects. γ-adducin null RBCs appear normal on Wright's stained peripheral blood smears and by scanning electron microscopy (SEM). The RBC count, hemoglobin content, hematocrit, MCV, reticulocyte %, osmotic fragility, and all other hematopoietic parameters are normal in γ-adducin null mice vs. wildtype. The apparent compensation by γ-adducin in β-adducin null red cells previously observed was tested by intercrossing mice null for γ- and β-adducin to produce βγ null double homozygotes. The additional loss of γ-adducin did not exacerbate the β-adducin null RBC phenotype as judged by examination of peripheral blood smears and SEM. Moreover, RBC osmotic fragility and complete blood counts in βγ-adducin null mice did not differ from β-adducin null mice. Western blotting of RBC ghost proteins confirmed reduction of α-adducin to ∼20% of normal in β-adducin null mice, as previously described. Strikingly, α-adducin in βγ-null RBC ghosts is reduced to barely detectable levels (<5% of normal). These studies show that (1) loss of γ-adducin alone does not significantly impact RBC membrane skeleton structure and function; (2) α- and β-adducin are stable and present at normal levels in the absence of γ-adducin; (3) the loss of γ-adducin in β-adducin null mice does not further exacerbate the β-adducin null HS phenotype; (4) the exacerbated loss of α-adducin in βγ double null RBCs suggests that up-regulated γ-adducin in β-adducin null mice associates in some way with and stabilizes α-adducin in the RBC membrane, but is unable to compensate functionally for the loss of the β subunit. We conclude that the normal function and stable incorporation of adducin into the peripheral membrane skeleton of red cells requires the presence of heterologous αβ binding subunits. Additional studies of adducin null mouse models, including our recently generated α-adducin null strain, will be useful tools in defining adducin functions and interactions in multiple tissues and organs.

Proteomic Analysis of RBC Ghosts from the Beta-Adducin and Protein 4.2 Knock-Out Mouse Models of Inherited Hemolytic Anemia.

Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. While it is known that mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability and non-erythroid effects of known RBC membrane skeleton mutations. To identify proteins that may be involved in disease severity and secondary effects, we used shotgun proteomics to globally profile proteins in RBC ghosts (i.e., RBC membrane skeleton and associated proteins) from well-defined mouse models of inherited hemolytic anemia. A peptide level ‘bottom-up' analysis was performed on RBCs from normal mice, beta-adducin knock-out mice (Add2-KO, compensated anemia), and protein 4.2 knock-out mice (4.2-KO, mild anemia). For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, five replicate runs utilizing 0.1 ug digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Normal versus diseased comparisons were made using a protein profile found consistently across all independent samples for each genotype. In total, 435 unique proteins were identified for the normal mouse RBC ghost. In contrast, 731 and 848 unique proteins were identified for the Add2-KO and 4.2-KO mice RBC ghosts respectively. Previously identified membrane skeleton proteins were found for all three genotypes with the predicted absence of the knock-out proteins. In addition to well-known membrane proteins, a surprising number of proteins were found involved in processes such as protein repair, protein degradation, Ras oncogene biology, and glycolysis. For both knock-out mice, a large number of proteins involved in translation were identified most likely reflecting their elevated reticulocytosis status. Comparison of the normal and Add2-KO RBC profiles revealed 5 proteins present only in normal the RBC while 53 proteins were present only in the diseased RBC. Likewise, normal vs 4.2 KO comparison revealed 6 proteins present only in the normal RBC while 111 were only in the diseased RBC. Comparison between the two KO mice revealed 34 proteins present only in the Add2-KO and 88 proteins present only in the 4.2 KO. Some of the identified differences are proteins with unknown functions (example, SH3-binding domain glutamic acid-rich protein like). Other differences involve proteins associated with diverse processes such as protein folding (Bcl2-associated athanogene 2), protein modification (magnesium-dependent phosphatase-1), protein transport (RAB35), metabolism (N-acetylneuraminic acid phosphatase), signal transduction (Prohibitin 2), and apoptosis (Rho GTPase activating protein 1). We report that tandem mass spec analysis of disease model RBC ghosts have demonstrated differences in their proteomes and that these identified differences potentially represent candidate proteins involved in disease severity and secondary effects.

Dysregulation of Phosphatidylserine Scrambling by Oxidative Stress.

An abnormal population of red blood cells (RBC) that exposes phosphatidylserine (PS) has been found in patients with sickle cell anemia and thalassemia. Enhanced formation of PS-exposing RBC results in their premature demise as these cells are removed from the circulation, and contributes to disease complications in the case that these cells are insufficiently removed. We hypothesize that increased oxidative stress, a common indication in these RBC, contributes to increased PS exposure. To evaluate the underlying mechanism, we compared the PS scrambling rate in three different mouse strains that sustain oxidative stress to their RBC: sickle mice, thalassemic mice (C57/Hbb[th3]), and mice deficient in peroxiredoxin II (prdx −/−). Oxidative damage is prominent in sickle and thalassemic RBC because of the presence of damaged hemoglobin and free heme components, and in prdx −/− mice because they lack a key component of oxidative damage repair. We determined the PS scrambling rate resulting from loading RBC with 100 microM intracellular Ca2+, in presence of either NEM (sulfhydryl alkylation), PDA (sulfhydryl modification), vanadate (flippase/ATPase inhibitor), or oligomycin (scrambling stimulation). As previously reported, sulfhydryl modifications cause flippase inhibition, and also change the PS scrambling rate with NEM treatment stimulating scrambling but PDA treatment reducing it. Sickle cells showed a 10% reduced susceptibility to NEM modification and no susceptibility to PDA, indicating shielding of the scramblase sulfhydryl groups. Thalassemic and Prdx −/− RBC showed a 30% and 10% reduced response to NEM modification, respectively, but normal reaction with PDA. The prdx −/− RBC contain a subpopulation that is severely oxidized as detected by an increase in autofluorescence. This cell population had a 10–15% lower susceptibility to NEM and PDA compared to the remaining population. The maximum percentage of PS-exposing cells (with oligomycin) was 10–20% reduced in all transgenic strains compared to normal, and even further reduced in the highly oxidized prdx−/− cells, indicating that a subset of cells had lost its ability to expose PS altogether. Yet, the baseline scrambling rate with 100 microM Ca2+ was increased 3-fold in thalassemic RBC, 1.5-fold in sickle mice, and 3-fold in the highly oxidized prdx −/− cells compared to normal mouse RBC, confirming the hypothesis that increased oxidative damage results in a higher propensity to scramble. Vanadate treatment had a stimulating effect on mouse RBC scrambling resulting in similar PS exposure in all mouse strains. While flippase activity was reduced in sickle RBC, this was not the case in thalassemic and prdx −/− RBC. In all RBC, increased scrambling activity resembled alterations similar to those seen after NEM treatment. Our data show that a subpopulation of oxidatively damaged cells is more prone to PS exposure during Ca2+ influx due to sulfhydryl modification. In addition, some oxidatively damaged cells lose their ability for Ca-induced scrambling. Together, we conclude that scrambling is dysregulated in oxidatively challenged RBC.

Phosphatidylserine Scrambling Is Faster in Sickle Mouse Erythrocytes Because of Oxidative Damage.

A subset of sickle red blood cells shows phosphatidylserine (PS) exposure on the red blood cell (RBC) surface. These cells contribute to the morbidity of the disease by increasing the patient's risk for stroke, altering the adhesive properties of the RBC, and affecting RBC survival. The cause and mechanism of production of these cells has not yet been resolved. PS asymmetry is normally maintained by activity of the flippase, which was shown to be inactivated in the PS-exposing sickle cells. Oxidative damage inside the sickle cells seems to contribute to this inactivation. PS exposure requires that phospholipids are randomized across the membrane by the Ca++-activated phospholipid scramblase. Thus far it has been unclear how small and transient increases (<100 nM) in intracellular Ca++ attained during sickle cell deoxygenation could facilitate this process. We now show that subtle oxidative modifications to the sulfhydryl groups on the scramblase can regulate its activity and lower its requirement for Ca++ to levels that might be reached in sickle cells. We evaluated the phospholipid scrambling rate, monitored by the appearance of PS-exposing cells over time, in sickle mouse (Berkeley type) and normal mouse RBC after loading the cells with Ca++ using ionophore. The level of intracellular Ca++ needed for scrambling was found to be at least 10 μM in normal mouse and human RBC and reached a maximum at 100 μM. Sickle cells scrambled at 1.5-fold the rate of normal cells when loaded with the same level of Ca++. Flippase inhibition by vanadate increased the scrambling rate both in normal and sickle cells, but did not lead to immediate scrambling in all cells and did not reduce the requirement for Ca++, suggesting that scramblase activation rather than flippase inhibition is the rate-limiting step in PS exposure. The sulfhydryl alkylating reagent n-ethylmaleimide (NEM) inhibits the flippase and enhances scrambling in human RBC, probably by changing the scramblase into an active conformation. This effect was similar in mouse RBC, leading to a markedly enhanced scrambling rate with more than 70% of the cells exposing PS after 10 minutes incubation in presence of 100 μM Ca++. In addition, we found that NEM treatment largely reduces the requirement for Ca++ with scrambling already effective below 1 μM. On the other hand, a similar compound, pyridyldithioethylamine (PDA), suppressed the scrambling process in normal mouse and human RBC by 50–80% even though it inhibits the flippase, showing that the scramblase can also be down-regulated by a different type of sulfhydryl modification. Interestingly, mouse sickle cells were not susceptible to inhibition by PDA, indicating altered sulfhydryl moieties on their scramblase. While most mouse sickle cells scrambled rapidly after NEM treatment similar to normal mouse cells, about 10% of the sickle cells were not responsive to NEM, also indicating previous sulfhydryl modifications. In conclusion, we show that the scramblase in mouse sickle cells is altered by sulfhydryl modifications, that could set up the sickle cells to expose PS more rapidly at lower intracellular Ca++ levels than normal RBC, leading to enhanced formation of PS-exposing cells in sickle cell disease.

Erythrocyte adhesion is modified by alterations in cellular tonicity and volume

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.

Oxidative Modifications to Flippase and Scramblase Affect Phosphatidylserine Exposure in Mouse Sickle Cells.

A subset of sickle red blood cells shows phosphatidylserine (PS) exposure on the red blood cell (RBC) surface. PS exposure has consequences for sickle cell pathophysiology, including an imbalance in hemostasis, enhanced cell recognition and adhesion to the endothelium. PS-exposing RBC are found among young reticulocytes as well as old dense sickle cells, but it is poorly understood how the PS-exposing cells are formed. PS exposure occurs when phospholipids are randomized across the membrane by the calcium-activated phospholipid scramblase. In addition, the flippase, which is responsible for transporting PS from the outer to the inner monolayer, needs to be inactivated. Indeed, we have previously found that all highly PS-exposing sickle cells have lost their flippase activity. We hypothesized then that transient calcium influx in RBC without flippase activity would result in PS exposure in sickle cells. However, brief calcium influx in normal red cells is not sufficient to induce PS exposure. Here we show that the scramblase activity is also altered by oxidative damage in sickle cells, which contributes to a higher susceptibility to calcium-induced PS exposure. We modified the sulfhydryl moieties of the flippase and the scramblase to see their respective effect on PS scrambling in sickle mouse (Berkeley type) and normal mouse RBC. We evaluated the scrambling rate by monitoring the appearance of PS-exposing cells over time after loading the cells with 0.1 mM calcium using ionophore. Sickle cells scrambled at twice the rate of normal cells when loaded with the same level of calcium. This could be accounted for by reduced flippase activity, since flippase inhibition by vanadate increased the formation of PS-exposing cells by 2.5-fold in normal mouse cells. However, a similar stimulation was also seen in sickle cells, indicating that not only flippase inhibition, but also enhanced scrambling contributes to PS exposure in sickle cells. The sulfhydryl alkylating reagent n-ethylmaleimide (NEM) inhibits the flippase and enhances scrambling in human RBC, probably by changing the scramblase into an active conformation. This effect was similar in mouse RBC, leading to a markedly enhanced scrambling rate with more than 70% of the cells exposing PS after 10 minutes incubation. While most sickle cells also scrambled rapidly after NEM treatment, about 20% of mouse sickle cells had lost their ability to scramble. Another sulfhydryl modifying reagent, pyridyldithioethylamine (PDA), suppresses the scrambling process in human RBC even though it inhibits the flippase, indicating a different type of sulfhydryl modification of the scramblase. Mouse RBC were slightly less susceptible to PDA than human cells, but the observed scrambling resulted in a lower degree of PS exposure per cell than normally found. Sickle cells, on the other hand, doubled their scrambling rate in presence of PDA, showing that the inhibiting effect on the scramblase was inactivated in these cells. In conclusion, mouse sickle cells show a higher rate of scrambling than normal RBC at the same level of intracellular calcium, although it is suppressed in a subpopulation. Sulfhydryl modification of the scramblase at the site that can be modified by PDA could prevent its downregulation and increase its susceptibility to PS exposure in response to transient calcium influx. Our data show that both flippase and scramblase sustain damage by oxidative stress in sickle cells.

Electroinsertion of xeno proteins in red blood cell membranes yields a long lived protein carrier in circulation

Electroinsertion, a novel method of implanting xeno-proteins in red blood cell plasma membranes is applied to the insertion of human glycophorin in mouse red blood cells. The procedures yield erythrocytes with approx. 10 4 glycophorin molecules per cell, displaying the patching phenomenon when reated with antiglycophorin monoclonal antibodies. Insertion of FITC-labeled glycophorin and subsequent quenching of FITC fluorescence with anti FITC antibody indicated that 70% of the inserted molecules were ‘correctly’ oriented, displaying the epitopes shown by glycophorin in human red blood cells. Moreover, insertion of FITC glycophorin in the red blood cell membranes yielded, under the fluorescence microscope continuous fluorescence which became patchy after reaction with anti-glycophorin monoclonal antibodies. When injected in mice, biotinylated RBC-Glyc + were shown to have the same life-span as normal mouse red blood cells, i.e. t 1/2 ≈ 12 days. Cytofluorometric assay of human glycophorin inserted in the red blood cell showed the same life-span for the inserted protein as for the red blood cell, with the protein fully ‘functional’, i.e. capable of binding antibodies. Human glycophorin inserted in mouse erythrocyte did not elicit any immune response in mice, whereas the same concentrations of free glycophorin injected i.v. were highly immunogenic.

IgM anti-erythrocyte autoantibodies specific for buried and neo-antigens using cellular-ELISA assays

The aim was to develop a cellular-ELISA assay to detect natural autoantibodies specific for bromelain-treated mouse red blood cells (BrMRBC). High, unexpected IgM titres against normal mouse red blood cells (NMRBC) were detected in day 7–14 sera of CBA mice treated with E. coli lipo-polysaccharide (LPS). These “autoantibodies” bound to normal mouse red blood cells in the presence or absence of commonly used c-ELISA adhering agents. Such high reactivity to NMRBC was never detected using complement dependent haemolytic assays in earlier work in this system. The question whether these IgM α-NMRBC molecules were binding nonspecifically (via Fc) or specifically (via Fab) was answered indirectly by comparing the binding titres of LPS-stimulated serum and several purified IgM antibody preparations (α-PC,α-KLH,MOPC 104E) on the same antigen coated plates. The observed binding ratios (titre on antigen X: titre on NMRBC) varied widely between different antibody sources, indicative of specific binding. In addition no significant unequivocal binding against NMRBC could be detected in vivo (LPS-stimulated mice) nor could bound IgM antibody be detected in a suspension-c-ELISA assay (high binding titres to BrMRBC could be detected in the latter test system). In conventional c-ELISA assays, modification of normal erythrocyte by adhesion to plastic microtitre plates appears to expose or create “neoantigens” on NMRBC which are not encountered in suspension-type c-ELISA, nor in lytic or agglutination assays where the erythrocyte targets are in suspension at physiological pH and isotonicity.

Evidence against the red blood cell origin of mitogenic factors in mouse malarial infection

The mean [ 3H]thymidine incorporation in response to stimulation of spleen cells of malaria-immune and non-immune BALB/c mice by normal mouse red blood cell culture supernatants were compared with unstimulated cultures of the same spleen cells. No significant difference was obtained between stimulated and unstimulated cultures for both immune and non-immune spleen cells. These findings do not support the hypothesis that erythrocyte-derived mitogenic factors occur in malarial infection.

Selective recognition in erythrophagocytosis by mouse peritoneal macrophages: Isolation and purification of a possible self-marker from mouse erythrocyte membranes

Rabbit anti-mouse red blood cells (RBC) antibody stimulated the phagocytosis of mouse RBC by isologous macrophages in two ways; mouse RBC pretreated with the antibody are susceptible to phagocytosis by isologous macrophages, while mouse macrophages pretreated with the antibody actively phagocytize even normal mouse RBC. The activity of the antibody to stimulate phagocytosis of isologous RBC, which can be called the opsonin activity, is specific for mouse and rat cells, since it was shown that the antibody could be absorbed on both mouse and rat cells, but not on sheep or rabbit cells. Non-immune rabbit serum did not show any opsonin activity. These results indicate that an antigen common to mouse and rat cells is responsible for preventing these cells from phagocytosis by mouse macrophages. The surface antigen of mouse RBC membranes was isolated and purified using the opsonininhibitor activity against mouse macrophages as a criterion of the self-marker of mouse cell. The opsonin-inhibitor activity of mouse RBC membranes was recovered as a lipoprotein in a fraction insoluble in 0.4 N-acetate, but soluble in 75% ethanol. This lipoprotein contained 81% lipid and 15% protein on a dry weight basis. Both the lipid and protein were indispensable for the activity of the opsonin-inhibitor. The opsonin-inhibitor activity against mouse macrophages was found only in fractions from mouse and rat RBC, not in similar fractions from sheep or rabbit RBC. This species-specificity seems to be due to the protein moiety, not the lipid moiety, since the latter was exchangeable with those of heterologous RBC fractions while the protein moiety was not.