Survival and senescence of human young red cells in vitro.

BACKGROUND: A number of experimental investigations in vivo suggest that in humans a decrease of circulating erythrocyte number ensues whenever erythropoietin (EPO) plasma level decreases. Since the process seems to selectively eliminate young red cells (neocytes), it has been named neocytolysis. The experimental models in vivo have revealed and documented multiple forms of neocytolysis but have not fully elucidated the specificity of the target red cells and the relation with EPO level changes. In an attempt to better characterize the neocytolytic process, we have undertaken an in vitro investigation on age-ranked human red cells. METHODS: By centrifugation on Percoll density gradient we separated the red cells population into three subsets, neocytes, middle-aged and old. Then we comparatively investigated the kinetics of survival of the subsets cultured under different conditions: with medium alone, with 10% autologous plasma, with EPO, alone or in combination with autologous monocytes. RESULTS: Neocytes showed a viability and a survival rate lower than the other red cells when cultured in medium or with 10% plasma. EPO at physiological doses increased their survival rate, but not that of the other subsets. This effect was enhanced by co-culture with monocytes. CONCLUSION: Likely neocytes are more sensitive than the other RBCs subsets to presence or absence of survival signals, such as EPO or plasma or monocytes derived factors. These observations could provide an insight into the link between the decrease in EPO plasma level and the reduction of circulating red cells mass and account for the specificity of neocytes clearance.

Neocytolysis: none, one or many? A reappraisal and future perspectives.

Neocytolysis is the hypothesis formulated to explain experimental evidence of selective lysis of young red blood cells (RBCs) (neocytes) associated with decreased plasma levels of erythropoietin (EPO). In humans, it appears to take place whenever a fast RBC mass reduction is required, i.e., in astronauts during the first days of spaceflight under weightlessness, where a fast reduction in plasma volume and increase in haematocrit occur. EPO plasma levels then decline and a decrease in RBC mass takes place, apparently because of the selective lysis of the youngest, recently generated RBCs (neocytes). The same process seems to occur in people descending to sea level after acclimatization at high altitude. After descent, the polycythaemia developed at high altitude must be abrogated, and a rapid reduction in the number of circulating RBCs is obtained by a decrease in EPO synthesis and the lysis of what seem to be young RBCs. In vivo, neocytolysis seems to be abolished by EPO administration. More recent research has ascribed to neocytolysis the RBC destruction that occurs under such disparate pathophysiologic conditions as nephropathy, severe obstructive pulmonary disease, blood doping, and even malaria anaemia. According to the theory, EPO's central role would be not only to stimulate the production of new RBCs in conditions of anaemia, as maintained by the orthodox view, but also that of a cytoprotective factor for circulating young RBCs. Why neocytes are specifically destroyed and how is this related to decreased EPO levels has not yet been elucidated. Changes in membrane molecules of young RBCs isolated from astronauts or mountain climbers upon return to normal conditions seem to indicate a higher susceptibility of neocytes to ingestion by macrophages. By limiting the context to space missions and high altitude expeditions, this review will address unresolved and critical issues that in our opinion have not been sufficiently highlighted in previous works.

Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: a study with bio-orthogonal chemical probes.

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton.

Expression of fetal hemoglobin in adult humans exposed to high altitude hypoxia.

In humans, acute erythroid expansion can lead to maturation of red blood cell (RBC) precursors containing fetal hemoglobin (F red cells). This can occur in patients after recovery from bone marrow transplantation, or in individuals affected by sickle cell or thalassemic syndromes. An accelerated erythroid lineage expansion is also a hallmark of the adaptive response to high altitude hypoxia. To explore the possible effect of this environment on F red cell production, we analyzed RBCs from five subjects during and after 17 days spent at high altitude and investigated the expression of fetal hemoglobin by different methodological approaches. By flow cytometry, we found a moderate increase of circulating F red cells during and after the hypoxia exposure, with respect to control cells analyzed before a stay at high altitude. The increased expression of γ-globin (as the specific subunit contained in F hemoglobin together with α-globin) was further confirmed by immunoblotting of young RBC hemolysates and quantitative RT-PCR of transcripts purified from a reticulocyte-enriched RBC fraction. Thus, in healthy adults the exposure to high altitude hypoxia induces maturation of F red cells at a level higher than under normal condition. The effect appears reduced after return to normoxia.

Application of gelatin zymography for evaluating low levels of contaminating neutrophils in red blood cell samples.

Supposedly "homogeneous" red blood cell (RBC) samples are commonly obtained by "washing" whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible artifactual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/106 RBCs.

Fragmentation of human erythrocyte actin following exposure to hypoxia.

In a comparative study on erythrocytes (RBCs) drawn from mountaineers before and after a high-altitude stay, we observed that upon returning to sea level, their RBCs displayed a senescent-like phenotype as indicated by their density and the partial loss of membrane proteins which are shed by ageing RBCs. The aim of this study was to investigate possible changes in the membrane skeleton of these RBCs and to compare them with pathological RBCs. We analysed the proteins of RBC ghosts obtained from our subjects before and after returning to sea level by two-dimensional electrophoresis and mass spectrometry. We observed lower expression and fragmentation of beta-actin after exposure to hypoxia. This suggested an alteration in membrane skeleton structure, which was confirmed by beta-actin release in cell lysates during ghost preparation. We observed a similar actin fragmentation and release in RBC lysates from beta-thalassaemic patients. In conclusion, these results indicate that after exposure to hypoxia, RBCs display a modification of their actin and cytoskeleton instability.

Red blood cell senescence and neocytolysis in humans after high altitude acclimatization.

A selective lysis of relatively young erythrocytes (neocytolysis), together with a decrease of erythropoietin (EPO) production, has been described in polycythemic, high altitude acclimatized climbers, after descent to sea level, and in astronauts, soon after exposure to weightlessness (Alfrey CP, Rice L, Udden MM, Driscoll TB. Neocytolysis may represent the physiological down-regulation of red-cell mass. Lancet 349 (1997) 1389-90). To study neocytolysis, we analysed blood samples drawn from 4 mountain climbers at sea level before and after 53 days of high altitude acclimatization (> or = 4500 m). After a 6-day descent to sea level, erythropoietin (EPO) plasma levels were lower than before high altitude acclimatization (mean values: 2.5+/-3.3 versus 10+/-4.5 mIU/ml, p < 0.05). Red blood cell (RBC) populations were separated into low, middle and high density subsets, which, by physical and phenotypical criteria, were characterized as young, middle-aged and old. RBC membrane molecules CD55 and CD59 along with phosphatydylserine and CD47 were measured. The former are partially lost during RBC aging. The latter are involved in the triggering or inhibition of RBC phagocytosis by macrophages. Immunofluorescence and flow cytometry were done on each density subset. Young and middle-aged RBCs largely disappeared after descent from high altitude (from 4.50% (+/-3.10) and 66% (+/-6.90) to 0.19% (+/-0.07) and 1.90% (+/-0.50), respectively). Simultaneously, there was a dramatic increase of high density RBCs (from 29.50% (+/-7) to 97.90% (+/-2.00)). Furthermore, the remaining young and middle-aged RBCs had acquired a senescent-like phenotype, which may account for their increased susceptibility to phagocytosis.

Activation of human T lymphocytes under conditions similar to those that occur during exposure to microgravity: a proteomics study.

A number of experiments, conducted under microgravity conditions, i.e. in space shuttle biolaboratories or in ground based systems simulating the conditions occurring in microgravity, show that in hypogravity, in vitro human lymphocyte activation is severely impaired. However, very early stimulation steps of T lymphocytes are not compromised, since CD69 receptor, the earliest membrane activation marker, is expressed by T cells at a level comparable to that observed on 1 g activated lymphocytes. Since CD69 engagement, together with submitogenic doses of phorbol esters, transduces an activation signal to T lymphocytes, we undertook a comparative study on the stimulation mediated through this receptor on human CD3+ cells cultured under conditions similar to those which occur during exposure to microgravity, i.e. in clinorotation, or at 1 g. During the early hours of activation, increased levels of intracellular calcium and increased mitochondrial membrane potential were detectable in clinorotating as well as in 1 g cells. However, after 48 hours clinorotation, interleukin 2 production by T lymphocytes was significantly reduced and cell proliferation was greatly decreased. By means of a differential proteomics approach on T cells activated in clinorotation or at 1 g for 48 hours, we were able to detect statistically significant quantitative protein alterations. Seven proteins with modified expression values were identified; they are involved in nucleic acids processing, proteasome regulation and cytoskeleton structure.

BMAP-28, an antibiotic peptide of innate immunity, induces cell death through opening of the mitochondrial permeability transition pore.

BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca(2+) and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.

Antigene effect in K562 cells of a PEG-conjugated triplex-forming oligonucleotide targeted to the bcr/abl oncogene.

Triplex-forming oligonucleotides are able to modulate gene expression by site-specific binding to genomic DNA. Their use as therapeutic agents is limited by inefficient cellular uptake, scarce nuclear internalization, and oligonucleotide self-aggregation. In this study, we demonstrate that a 13-mer AG motif oligonucleotide covalently linked to a high-molecular mass (9000 Da) polyethylene glycol (PEG ODN(13)) exhibits uptake and biological properties that are superior to those of the nonconjugated isosequence analogue (free ODN(13)). Band-shift and footprinting experiments showed that PEG ODN(13) forms a stable triple helix (apparent K(d) between 10(-6) and 10(-7) M in 50 mM Tris-acetate, 10 mM MgCl(2), pH 7.4, 37 degrees C) with a natural polypurine-polypyrimidine target located in the 5' flanking region of the human bcr/abl oncogene. Confocal laser microscopy performed on unfixed live cells stained with hexidium iodide as well as on glass-fixed cells stained with propidium iodide showed that fluorescein-labeled PEG ODN(13) is far more efficiently taken up and internalized in the nucleus by K562 and HeLa cells than the nonconjugated free ODN(13). It was found that PEG ODN(13) specifically downregulated the transcription of bcr/abl mRNA at 65 +/- 5% with respect to control and inhibited cell growth by 32 +/- 3% in a 3 day liquid culture assay. Moreover, PEG ODN(13) was more resistant against S1 and fetal bovine serum nucleases than free ODN(13), and less inclined to self-associate into multistrand structures in solution. Taken together, these results provide useful elements for designing artificial transcription repressors with enhanced potency in vivo.