Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

Lysophosphatidic acid-induced pro-thrombotic phosphatidylserine exposure and ionophore-induced microvesiculation is mediated by the scramblase TMEM16F in erythrocytes.

Recent studies indicate that erythrocytes actively modulate blood clotting and thrombus formation. The lipid mediator lysophosphatidic acid (LPA) is produced by activated platelets, and triggers a signaling process in erythrocytes. This results in cellular calcium uptake and exposure of phosphatidylserine (PS) at the cell surface, thereby generating activated membrane binding sites for factors of the clotting cascade. Moreover, erythrocytes of patients with a bleeding disorder and mutations in the scramblase TMEM16F show impaired PS exposure and microvesiculation upon treatment with calcium ionophore. We report that TMEM16F inhibitors tannic acid (TA) and epigallocatechin-3-gallate (EGCG) inhibit LPA-induced PS exposure and calcium uptake at low micromolar concentrations; fluoxetine, an antidepressant and a known activator of TMEM16F, enhances these processes. These effectors likewise modulate erythrocyte PS exposure and microvesicle shedding induced by calcium ionophore treatment. Further, LPA-treated erythrocytes triggered thrombin generation in platelet-free plasma which was partially impaired in the presence of TA and EGCG. Thus, this study suggests that LPA activates the scramblase TMEM16F in erythrocytes, thereby possibly mediating a pro-thrombotic function in these cells. EGCG as well as fluoxetine, substances with potentially high plasma concentrations due to alimentation or medical treatment, should be considered as potential effectors of systemic hemostatic regulation.

Glycated hemoglobin concentrations of red blood cells minimally increase during storage under standard blood banking conditions.

BACKGROUND: Few and inconsistent data exist describing the effect of storage duration on glycated hemoglobin (HbA1c) concentrations of red blood cells (RBCs), impeding interpretation of HbA1c values in transfused diabetic patients. Hence the aim of this study was to evaluate to what extent HbA1c concentrations of RBCs change during the maximum allowed storage period of 42 days. STUDY DESIGN AND METHODS: Blood was drawn from 16 volunteers, leukofiltered, and stored under standard blood banking conditions. HbA1c concentrations of RBCs were measured on Days 1 and 42 of storage using three different validated devices (ion-exchange high-performance liquid chromatography Method A1 and A2, turbidimetric immunoassay Method B). RESULTS: Mean HbA1c concentrations of RBCs on Day 1 were 5.3 ± 0.3% (Method A1), 5.4 ± 0.4% (Method A2), and 5.1 ± 0.4% (Method B). HbA1c concentrations increased to 5.6 ± 0.3% (A1, p < 0.0001), 5.7 ± 0.3% (A2, p = 0.004), and 5.5 ± 0.4% (B, p < 0.0001) on Day 42, respectively, corresponding to a 1.06-fold increase across all methods. Glucose concentrations in the storage solution of RBCs decreased from 495 ± 27 to 225 ± 55 mg/dL (p < 0.0001), confirming that stored RBCs were metabolically active. CONCLUSION: These results suggest a significant, albeit minor, and most likely clinically insignificant increase in HbA1c concentrations during storage of RBCs for 42 days.

Drug-induced endovesiculation of erythrocytes is modulated by the dynamics in the cytoskeleton/membrane interaction.

Recent studies on erythrocyte membrane fluctuations revealed that the erythrocyte cytoskeleton actively modulates its membrane association thereby regulating crucial membrane properties. Cationic amphiphilic drugs like chlorpromazine are known to induce a cup-like cell shape and vesicle formation into the cell interior, effectors of this process, however, are largely unknown. Using flow cytometry, this study explored conditions that influence endovesiculation induced by chlorpromazine. We found that inhibitors of membrane fluctuations, like ATP depletion, vanadate or fluoride, also inhibited endovesiculation whereas activation of PKC, known to decrease cytoskeleton association and increase membrane fluctuations, also enhanced endovesicle formation. This indicates that endovesicle formation and membrane fluctuations are modulated by the same cytoskeleton-regulated membrane properties. Further, acanthocytic erythrocytes of chorea acanthocytosis (ChAc) patients that lack the VPS13A/chorein protein - likely a crucial organizer at the erythrocyte cytoskeleton/membrane interface - showed a strong decrease in chlorpromazine-induced endovesiculation. The responses of ChAc erythrocytes to effectors of endovesiculation were similar to that of control erythrocytes, yet at drastically reduced levels. This suggests a more rigid and less dynamic interaction at the membrane-cytoskeleton interphase of ChAc erythrocytes.

Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation.

Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.

Storage of packed red blood cells impairs an inherent coagulation property of erythrocytes.

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process.

A Potential Citrate Shunt in Erythrocytes of PKAN Patients Caused by Mutations in Pantothenate Kinase 2.

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography-mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation.

Packed red blood cell transfusion in preterm infants.

Premature infants commonly receive adult packed red blood cells (pRBCs) during their hospital stay. As adult erythrocytes differ substantially from those of preterm infants, transfusion of adult pRBCs into preterm infants can be considered inappropriate for the physiology of a preterm infant. An absence of standardisation of transfusion protocols makes it difficult to compare and interpret pertinent clinical data, as reflected by unclear associations between pRBC transfusion and complications related to prematurity, such as bronchopulmonary dysplasia, neurodevelopmental impairment, retinopathy of prematurity, or necrotising enterocolitis. The difficulty in interpreting clinical data is further increased by differences in study designs that either overestimate pRBC-associated complications of prematurity or have not yet been designed to directly link pRBC transfusions to their respective complications. Thus, neonatal transfusion practice has become an ongoing difficulty, in which differences in transfusion guidelines hinder the ability to generate comparable clinical data, and heterogeneity in clinical data prevents the implementation of standardised transfusion protocols. To overcome these issues, novel approaches with biochemical-clinical translational designs could enable clinicians to gather causal evidence instead of circumstantial correlation.

Evidence for a size-sensing mechanism in animal cells.

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.

Redox Properties of Human Erythrocytes Are Adapted for Vitamin C Recycling.

Ascorbic acid (AA; or vitamin C) is an important physiological antioxidant and radical scavenger. Some mammalian species, including homo sapiens, have lost the ability to synthetize AA and depend on its nutritional uptake. Erythrocytes from AA-auxotroph mammals express high amounts of the glucose transporter GLUT1. This isoform enables rapid uptake of glucose as well as dehydroascorbate (DHA), the fully oxidized form of AA. Here, we explored the effects of DHA uptake on the redox metabolism of human erythrocytes. DHA uptake enhanced plasma membrane electron transport (PMET) activity. This process is mediated by DCytb, a membrane bound cytochrome catalyzing extracellular reduction of Fe3+ and ascorbate free radical (AFR), the first oxidized form of AA. DHA uptake also decreased cellular radical oxygen species (ROS) levels. Both effects were massively enhanced in the presence of physiological glucose concentrations. Reduction of DHA to AA largely depleted intracellular glutathione (GSH) and induced the efflux of its oxidized form, GSSG. GSSG efflux could be inhibited by MK-571 (IC 50 = 5 µM), indicating involvement of multidrug resistance associated protein (MRP1/4). DHA-dependent GSH depletion and GSSG efflux were completely rescued in the presence of 5 mM glucose and, partially, by 2-deoxy-glucose (2-DG), respectively. These findings indicate that human erythrocytes are physiologically adapted to recycle AA both intracellularly via GLUT1-mediated DHA uptake and reduction and extracellularly via DCytb-mediated AFR reduction. We discuss the possibility that this improved erythrocyte-mediated AA recycling was a prerequisite for the emergence of AA auxotrophy which independently occurred at least twice during mammalian evolution.

Igbp1 is part of a positive feedback loop in stem cell factor-dependent, selective mRNA translation initiation inhibiting erythroid differentiation.

Stem cell factor (SCF)-induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (eIF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of eIF4E renders proliferation of erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and eIF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbp1), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbp1 impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple eIF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbp1 acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selective mRNA recruitment to polysomes in the balance between proliferation and maturation of erythroblasts.

Stat5 regulates cellular iron uptake of erythroid cells via IRP-2 and TfR-1.

Erythropoiesis strictly depends on signal transduction through the erythropoietin receptor (EpoR)-Janus kinase 2 (Jak2)-signal transducer and activator of transcription 5 (Stat5) axis, regulating proliferation, differentiation, and survival. The exact role of the transcription factor Stat5 in erythropoiesis remained puzzling, however, since the first Stat5-deficient mice carried a hypomorphic Stat5 allele, impeding full phenotypical analysis. Using mice completely lacking Stat5--displaying early lethality--we demonstrate that these animals suffer from microcytic anemia due to reduced expression of the antiapoptotic proteins Bcl-x(L) and Mcl-1 followed by enhanced apoptosis. Moreover, transferrin receptor-1 (TfR-1) cell surface levels on erythroid cells were decreased more than 2-fold on erythroid cells of Stat5(-/-) animals. This reduction could be attributed to reduced transcription of TfR-1 mRNA and iron regulatory protein 2 (IRP-2), the major translational regulator of TfR-1 mRNA stability in erythroid cells. Both genes were demonstrated to be direct transcriptional targets of Stat5. This establishes an unexpected mechanistic link between EpoR/Jak/Stat signaling and iron metabolism, processes absolutely essential for erythropoiesis and life.

Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2.

Erythropoiesis requires erythropoietin (Epo) and stem cell factor (SCF) signaling via their receptors EpoR and c-Kit. EpoR, like many other receptors involved in hematopoiesis, acts via the kinase Jak2. Deletion of EpoR or Janus kinase 2 (Jak2) causes embryonic lethality as a result of defective erythropoiesis. The contribution of distinct EpoR/Jak2-induced signaling pathways (mitogen-activated protein kinase, phosphatidylinositol 3-kinase, signal transducer and activator of transcription 5 [Stat5]) to functional erythropoiesis is incompletely understood. Here we demonstrate that expression of a constitutively activated Stat5a mutant (cS5) was sufficient to relieve the proliferation defect of Jak2(-/-) and EpoR(-/-) cells in an Epo-independent manner. In addition, tamoxifen-induced DNA binding of a Stat5a-estrogen receptor (ER)* fusion construct enabled erythropoiesis in the absence of Epo. Furthermore, c-Kit was able to enhance signaling through the Jak2-Stat5 axis, particularly in lymphoid and myeloid progenitors. Although abundance of hematopoietic stem cells was 2.5-fold reduced in Jak2(-/-) fetal livers, transplantation of Jak2(-/-)-cS5 fetal liver cells into irradiated mice gave rise to mature erythroid and myeloid cells of donor origin up to 6 months after transplantation. Cytokine- and c-Kit pathways do not function independently of each other in hematopoiesis but cooperate to attain full Jak2/Stat5 activation. In conclusion, activated Stat5 is a critical downstream effector of Jak2 in erythropoiesis/myelopoiesis, and Jak2 functionally links cytokine- with c-Kit-receptor tyrosine kinase signaling.

PKAN neurodegeneration and residual PANK2 activities in patient erythrocytes.

OBJECTIVE: Pantothenate kinase 2-associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration. METHODS: Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry-Albright Dystonia Scale (BAD-Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level. RESULTS: Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12-56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations. INTERPRETATION: Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient-derived cell system with still underestimated diagnostic potential.

The different functions of Stat5 and chromatin alteration through Stat5 proteins.

Stat5 proteins modulate gene transcription upon cytokine- and growth factor action. Stat5a and Stat5b proteins alone are weak activators of transcription. They can modify chromatin organization through oligomerization and they act predominantly in co-operation and interaction with other proteins. The conservative view of exclusively nuclear functions of Stat5 was challenged by the observation of additional Stat5 effects in the cytoplasm, resulting in activation of the PI3K-Akt pathway. We summarize biological consequences of mutations in conserved domains of Stat5 or of deletions in the N- or C-terminal domains with impact on target gene transcription. Formation of higher-order oligomers is dramatically changed upon amino- or carboxyterminal deletions in Stat5 proteins. Mutations in or deletion of the Stat5 N-terminus leads to diminished leukemogenic potential of oncogenic Stat5, probably due to the inability to form Stat5 tetramers. The Stat5 N-terminal domain prevents persistent activation and can act as a DNA-docking platform for the glucocorticoid receptor (GR). The corresponding protocols should facilitate follow-up studies on Stat5 proteins and their contribution to normal physiological versus pathological processes through differential chromatin binding.

Apoptosis protection by the Epo target Bcl-X(L) allows factor-independent differentiation of primary erythroblasts.

BACKGROUND: Erythropoietin (Epo) is required for correct execution of the erythroid differentiation program. Erythropoiesis requires Bcl-X(L), a major late target of Epo-receptor signaling. Mice lacking Bcl-X(L) die around embryonic age E12.5, forming normal erythroid progenitors but lacking functional red cells. Recently, serum-free culture conditions for expansion of murine red cell progenitors were developed, yielding cells capable of in vivo-like terminal differentiation into enucleated erythrocytes, in response to Epo/insulin. Here we address whether Epo function during terminal maturation involves a cytokine-independent "default program," requiring only apoptosis inhibition through Epo-dependent upregulation of Bcl-X(L). RESULTS: Exogenous expression of Bcl-X(L) or Bcl-2 in primary murine erythroblasts or clonal erythroblast lines derived from p53(-/-) mice allowed these cells to undergo terminal erythroid maturation, in the complete absence of cytokines. A potential autocrine Epo loop was ruled out by respective neutralizing antibodies. Importantly, sustained proliferation of Bcl-X(L)-expressing immature erythroblasts still required respective factors (Epo, stem cell factor [SCF], and the glucocorticoid receptor ligand dexamethasone [Dex]). Epo-independent differentiation in these Bcl-X(L)- or Bcl-2-expressing, primary erythroblasts was thus triggered by removal of the renewal factors SCF and Dex. This initiated the maturation-specific expression cascade of erythroid transcription factors, followed by differentiation divisions (characterized by a short G1 phase and decrease in cell size), hemoglobin accumulation, and enucleation. CONCLUSIONS: During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X(L). This allows "default" terminal differentiation of apoptosis-protected, committed erythroblasts, independent of any exogenous signals.

Iron regulatory protein-independent regulation of ferritin synthesis by nitrogen monoxide.

The discovery of iron-responsive elements (IREs), along with the identification of iron regulatory proteins (IRP1, IRP2), has provided a molecular basis for our current understanding of the remarkable post-transcriptional regulation of intracellular iron homeostasis. In iron-depleted conditions, IRPs bind to IREs present in the 5'-UTR of ferritin mRNA and the 3'-UTR of transferrin receptor (TfR) mRNA. Such binding blocks the translation of ferritin, the iron storage protein, and stabilizes TfR mRNA, whereas the opposite scenario develops when iron in the intracellular transit pool is plentiful. Nitrogen monoxide (commonly designated nitric oxide; NO), a gaseous molecule involved in numerous functions, is known to affect cellular iron metabolism via the IRP/IRE system. We previously demonstrated that the oxidized form of NO, NO(+), causes IRP2 degradation that is associated with an increase in ferritin synthesis [Kim, S & Ponka, P (2002) Proc Natl Acad Sci USA99, 12214-12219]. Here we report that sodium nitroprusside (SNP), an NO(+) donor, causes a dramatic and rapid increase in ferritin synthesis that initially occurs without changes in the RNA-binding activities of IRPs. Moreover, we demonstrate that the translational efficiency of ferritin mRNA is significantly higher in cells treated with SNP compared with those incubated with ferric ammonium citrate, an iron donor. Importantly, we also provide definitive evidence that the iron moiety of SNP is not responsible for such changes. These results indicate that SNP-mediated increase in ferritin synthesis is, in part, due to an IRP-independent and NO(+)-dependent post-transcriptional, regulatory mechanism.