Daniela Bratosin

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span

Comparing the properties of ‘young’ and senescent (‘aged’) O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a β-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows:(1)Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes.(2)RCA120 as well asErythrina cristagalli andErythrina corallodendron lectins specific for terminal β-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation.(3)The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by α-2,6-sialyltransferase of fluorescent or radioactiveN-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively.(4)Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal β-galactose specific macrophage lectin.(5)Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to thein vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages

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, Neu5,9Ac2, Neu5,7Ac2, Neu (de‐N‐acetylated‐Neu5Ac), Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac2, Neu5,8Ac29Lt and Neu5Ac8S were characterised. Among these different compounds, Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac2, Neu5,8Ac29Lt 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.

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.

Molecular mechanisms of erythrophagocytosis: Flow cytometric quantitation of in vitro erythrocyte phagocytosis by macrophages

A rapid, sensitive, and reproducible flow cytofluorimetric procedure is described for quantitation of erythrophagocytosis based on the use of red blood cells (RBCs) labeled with the fluorescent probe PKH-26. The procedure involves the following steps: i) incubation of PKH-26-labeled erythrocytes with macrophages, ii) removal of un-bound red blood cells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macrophages. Each step was controlled by fluorescence microscopy. Use of fluorescent, instead of radio-labeled RBCs, makes the method more sensitive, rapid, and avoids radioactive hazards. Furthermore, this approach is multi-parametric and can distinguish different populations of macrophages with reference to their erythrophagocytic potential. This technology moreover, has broad applications from the initial step of contact (between effector and target cells to study the specific receptor mediated attachment) to the subsequent cascade of time-dependent changes resulting from that signal transduction.

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.

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.

Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?

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.

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.