A cytometric study of the red blood cells in Gaucher disease reveals their abnormal shape that may be involved in increased erythrophagocytosis.

BACKGROUND: Gaucher disease is a sphingolipidosis caused by a deficiency of the enzyme glucocerebrosidase. Macrophages transform into pathogenic Gaucher cells following the phagocytosis of red blood cells (RBCs) and subsequent accumulation of glucosylceramide. Enhanced erythrophagocytosis is one feature of the disease indicating abnormal macrophage-RBC interactions. We hypothesized that the erythrophagocytosis observed in Gaucher disease may be at least partly due to abnormalities in the RBCs themselves. METHODS: To investigate this hypothesis, we used flow cytometry FSC/SSC to study RBCs sampled from seven patients with Gaucher disease in terms of their shape and the expression of markers of senescence and phagocytosis. Cells from two of the seven patients were evaluated before and 9 months after the start of enzyme-replacement therapy. RESULTS: Untreated patients were found to have abnormal flow-cytometry profiles suggesting an alteration of Gaucher RBC morphology. Scanning electron microscopy confirmed this finding by revealing many abnormally shaped RBCs. Whereas there was no evidence of desialylation of membrane glycoconjugates or phosphatidylserine exposure, RBC viability (calcein-AM test) and CD47 expression were reduced. These anomalies found in RBCs sampled from two patients before treatment, were no longer present after a 9 month-long enzyme-replacement therapy. CONCLUSIONS: We report on previously overlooked alterations of Gaucher RBCs that may facilitate erythrophagocytosis in untreated patients. Their potential role in the anemia, the excess of aggregation and rheological anomalies associated with Gaucher disease must now be addressed. RBC anomalies may take part in the abnormal crosstalk between RBCs and macrophages leading to the accumulation of Gaucher cells.

Active caspases-8 and -3 in circulating human erythrocytes purified on immobilized annexin-V: a cytometric demonstration.

Human red blood cells (RBCs) have a normal life span of 120 days in vivo and might be primed in vitro to die in response to apoptotic stimuli through a caspase-independent pathway. It is well known that, in vivo, aging RBCs externalize phosphatidylserine residues but is unknown whether these cells express active caspases at this stage. We isolated RBCs expressing phosphatidylserine on their surface from human blood by applying an original method of affinity chromatography using annexin-V fixed on gelatin or on magnetic beads. The isolated RBCs were then analyzed by flow cytometry for morphological changes (dot-plot forward scatter versus side scatter), phosphatidylserine externalization (annexin-V test), cell viability (calcein-AM test), and caspase activities using fluorescent substrates specific for caspases-3 and -8. In addition, cells were systematically visualized using phase contrast, fluorescence, and confocal microscopy. We found that the population of RBCs fixed on annexin-V is a mixture of discocytes and shrunken cells. This annexin-V-positive population showed a dramatic loss of viability based on esterase activity determination (calcein-AM test). Moreover, we demonstrated that circulating RBCs express both active caspases-8 and -3 in half of the annexin-V-positive cells. All of these results were confirmed by phase contrast, fluorescence, and confocal microscopy. Our results demonstrate active caspases in RBC isolated from blood suggesting that caspases may participate in the regulation of in vivo RBC half-life. This finding open the door to fruitful investigations in the field of RBC pathology.

Pathogenesis of osteoarthritis: chondrocyte replicative senescence or apoptosis?

BACKGROUND: The aim of this study was to investigate by flow cytometry cellular viability and apoptosis of human chondrocytes isolated from osteoarthritic cartilage and to correlate replicative senescence with apoptosis of these cells. METHODS: To understand the mechanisms underlying the process of cell death in cartilage destruction, we investigated by flow cytometry cellular viability (Cell viability calcein-AM assay) and apoptosis (Light scattering properties of chondrocytes, study of chondrocyte death using Annexin-V-FITC and propidium iodide double-labeling, caspase-3 activity determination) of human chondrocytes isolated from osteoarthritic and nonosteoarthritic cartilage. Senescent cells were characterized using the senescence-associated-beta-galactosidase marker (SA-beta-Gal marker) by staining with chromogenic substrate (X-Gal) to produce blue coloration of SA-beta-Gal-positive cells and microscopy analysis. RESULTS: The results we obtained show that between 25 and 40% of chondrocytes were in apoptosis and all of them were SA-beta-Gal-positive. CONCLUSIONS: These results demonstrate that the death of osteoarthritic chondrocytes is an apoptotic phenomenon which is preceded by an accelerated mechanism of replicative senescence.

Reduced diversity of the human erythrocyte membrane sialic acids in polycythemia vera. Absence of N-glycolylneuraminic acid and characterisation of N-acetylneuraminic acid 1,7 lactone.

Sialic acids from the erythrocyte (RBC) membrane of a patient suffering from polycythemia vera, a malignant orphan disorder of hematopoietic cells, was studied using GC/MS. We found that the sialic acid diversity of these membranes was drastically reduced since only four entities were identified: Neu5Ac (91.5%) and its 1,7 lactone Neu5Ac1,7L (7.5%) which is absent in normal RBC, Neu4,5Ac(2) (0.50%) and Neu4,5Ac(2) 9Lt (0.50%); in normal RBC, Neu5,7Ac(2), Neu5,9Ac(2), Neu5Ac9Lt, Neu5Ac8S and Neu, as well as traces of Kdn, were also present. Neu5Gc and its O-alkylated or O-acetylated derivatives, which are considered by various authors as cancer markers, were not detected.

Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging.

BACKGROUND: A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported. METHODS: The assay described in this report is based on the use of acetoxymethyl ester of calcein (calcein-AM), a fluorescein derivative and nonfluorescent vital dye that passively crosses the cell membrane of viable cells and is converted by cytosolic esterases into green fluorescent calcein, which is retained by cells with intact membranes and inactive multidrug resistance protein. The loss of calcein can be easily determined by flow cytometry, and the cytosolic localization of esterases was demonstrated by spectrofluorometric analyses. RESULTS: We found that RBCs incubated with Ca(2+), which induces a rapid and modulated self-death that shares several features with apoptosis (Bratosin et al., Cell Death Differ 2001;8:1143-1156), externalized phosphatidylserine and lost calcein staining and cytosolic adenosine triphosphate content. Double labeling using phycoerythrin-labeled annexin-V and calcein-AM showed that the decrease of esterase activity is an early event that precedes the externalization of phosphatidylserine residues. In addition, this assay allowed us to distinguish young and aged RBCs isolated by ultracentrifugation in a self-forming Percoll gradient and can be considered as a reliable marker of RBC aging. CONCLUSIONS: Calcein-AM assay may represent a wide application for assessing RBC viability, particularly in blood banks.

On the evolution of erythrocyte programmed cell death: apoptosis of Rana esculenta nucleated red blood cells involves cysteine proteinase activation and mitochondrion permeabilization.

Batracian Rana esculenta erythrocytes cell death induced by either calcium influx, or staurosporine, involves typical apoptotic phenotype. Our data reveal: (i) a drastic modification of the cell morphology with loss of the ellipsoidal form as assessed by phase contrast microscopy and scanning electron microscopy; (ii) an exposure of the phosphatidylserine residues in the outer leaflet of the cell membrane; (iii) a caspase-3-like activity; (iv) a mitochondrial membrane potential (Delta Psi m) loss; and (v) a chromatin condensation and fragmentation. Erythrocyte chromatin condensation and fragmentation are prevented by caspase and calpain peptide inhibitors. These inhibitors also prevent Delta Psi m loss supporting the idea that mitochondria is a central sensor for Rana erythrocytes cell death. Our observations highlight the conservation of the programmed cell death machinery in erythrocytes across kingdom.

Uptake of sialic acid by human erythrocyte. Characterization of a transport system.

Upon incubation of human red blood cells (RBC) with [4-9-14C] N-acetylneuraminic acid, the cells incorporated this sugar, as demonstrated by the identification of labelled N-acetylmannosamine in the cytosol, as a result of the action of the sialic acid pyruvate-lyase we discovered previously (Biochimie 84 (2002) 655). The mechanism is saturable and indicates the presence of a limited number of transporter molecules in the RBC membrane. This transport process may have relevance to the desialylation of membrane glycoconjugates which occurs during ageing of erythrocytes.

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups.

The composition of the human erythrocyte membrane (RBC) glycoprotein- and glycolipid-bound sialic acids of A, B, AB and O type donors was studied using a new method (Zanetta et al., Glycobiology 11 (2001) 663-676). In addition to Neu5Ac as the major compound, Kdn, Neu5,9Ac(2), Neu5,7Ac(2), Neu (de-N-acetylated-Neu5Ac), Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac(2), Neu5,8Ac(2)9Lt and Neu5Ac8S were characterised. Among these different compounds, Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac(2), Neu5,8Ac(2)9Lt and Neu5Ac8S have never been described and quantitatively determined before in human tissues or cells. Neu5Gc and its O-alkylated or O-acylated derivatives were not detected.

Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes.

Sialate pyruvate-lyases, also known as sialate aldolases (EC 4.1.3.3), reversibly catalyse the cleavage of free N-acetylneuraminic acids to form pyruvate and N-acetylmannosamine. These enzymes are widely distributed and are present in numerous pro- and eukaryotic cells, in which they are localized only in the cytosol. They play an important role in the regulation of sialic acid metabolism by controlling the intracellular concentration of sialic acids of biosynthetic or exogenous origin, thus preventing the accumulation of toxic levels of this sugar. Application of an original colorimetric micromethod for N-acetylmannosamine determination, as well as the use of [4,5,6,7,8,9-14C]N-acetylneuraminic acid, led us to evidence a cytosolic neuraminate aldolase activity in human red blood cells (RBCs) and then to define the main characteristics of this enzyme: Michaelis-Menten type, K(m:) 1.4 +/- 0.05 mM, optimal pH: 7.6 +/- 0.2, optimal temperature: 70 +/- 2 degrees C, inhibition by heavy metals: Ag(+) and Hg(++). These enzyme parameters are close to those of the bacterial and mammalian aldolases described up to now. At the moment, the presence of sialate pyruvate-lyase in the cytosol of red blood cells remains an enigma.

Flow cytometric approach to the study of erythrophagocytosis: evidence for an alternative immunoglobulin-independent pathway in agammaglobulinemic mice.

Neuraminidase treatment of red blood cells (RBCs) is believed to induce changes similar to RBC senescence, and leads to a rapid clearance of RBCs from the circulation in vivo. The objective of this study using immunodeficient SCID mice and the lipophilic fluorescent probe PKH-26 was to ascertain whether antibodies are required as the final signal allowing the phagocytosis of neuraminidase-treated murine RBCs. All of the methods we applied are based on flow cytometry analysis using fluorescent probes: fluoresceinyl isothiocyanate (FITC)-labeled lectins for membrane carbohydrate identification and PKH-26-labeled RBCs for in vitro phagocytosis and in vivo clearance studies. The results can be summarized as follows: (i) the rate of neuraminidase-induced desialylation of RBCs from normal and immunodeficient mice is identical as ascertained with FITC-labeled lectins (wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA(120))); (ii) the rate of clearance of enzyme-treated RBCs from both types of mice is also similar, as is their localization in spleen, liver and lung; (iii) the rates of in vitro phagocytosis of untreated and neuraminidase-treated PKH-26-labeled RBCs from both species of mice are very similar in the presence of homologous sera. In the absence of serum or in the presence of heterologous sera, the rate of phagocytosis is markedly decreased but not totally abolished. These data suggest that neuraminidase-treated RBCs can be cleared via an alternative pathway that is antibody-independent. This pathway exists in immunocompetent mice but with a very low activity and is the only one active in immunodeficient mice. In accordance with results reported by Connor et al. [J. Biol. Chem. 269 (1994) 2399], it is possible that this antibody-independent mechanism is involved in the clearance of circulating senescent RBCs. Finally, the methods described here may also be of interest for the investigation of the mechanisms involved in the phagocytosis of apoptotic cells.