Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with⁵¹Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations⁵¹Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning⁵¹Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes. Studies by partitioning in two-polymer aqueous phases

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic 59Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.     

Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes: Studies by partitioning in two-polymer aqueous phases

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic ⁵⁹Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. II. A correction

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of human rosette- and nonrosette-forming lymphoid cells by countercurrent distribution in an aqueous two-phase system.

Mixtures of aqueous solutions of dextran and of poly(ethylene glycol) form immiscible two-phase systems suitable for the separation (by partition) of cells based on subtle differences in membrane surface properties. Lymphoid cells from human tonsils were subjected to countercurrent distribution in such a system. Analysis of cells from different parts of the countercurrent extraction train by the sheep red blood cell rosette test indicates a separation of rosette-forming and nonrosette-forming lymphocytes with the former having the higher partition coefficient. Since a major determinant of cell partition in these phases is a membrane-charge associated property it appears likely that the rosette-forming cells have a higher surface charge than the nonrosette-forming cells.     

Cell-cell affinity probed by countercurrent distribution in two-polymer aqueous phase systems

Aqueous solutions of dextran and of polyethylene glycol when mixed form immiscible liquid two-phase systems with a polyethylene glycol-rich top and a dextran-rich bottom. Such phases can be buffered and rendered isotonic and are suitable for the partition of cells. The partition coefficient of cells (i.e., their relative affinity for the top or bottom phase or their adsorption at the interface) depends on the polymer concentrations, on the ionic composition and concentration and, most sensitively, on their membrane surface properties. When two cell populations are mixed the partition coefficient of each is unaffected by the presence of the other population unless an interaction takes place between them. By countercurrent distribution of two cell populations, in a phase system selected such that the partition coefficient of one population is high (i.e., more cells in the top phase) and of the other low, one should be able to detect subtle interactions between cells in the two populations should they occur. Results of experiments with a model system consisting of human peripheral blood mononuclear cells and sheep red blood cells bear out the feasibility of this approach. At low ratios of sheep erythrocytes to mononuclear cells no interaction is apparent. With increasing ratios there is an increasing shift in the distribution curve of subpopulations of mononuclear cells (i.e., T-lymphocytes) as they interact with the sheep red cells. Substitution of rabbit red cells for sheep erythrocytes (at high ratios) reveals a small yet significant shift of a subpopulation of mononuclear cells as well. Distribution of human peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (all B-lymphocytes) are essentially unaffected by the presence of sheep red cells. This sensitive new method, still in its infancy, holds out the hope for the detection of previously unknown cell-cell affinities and for probing suspected cell-cell interactions.     

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.     

Effect of acetaldehyde and glutaraldehyde fixation on the surface properties of red blood cells as determined by partition in aqueous phases

A correlation between partition of cells in dextran/polyethylene glycol aqueous phase systems and their relative electrophoretic mobilities is often in evidence. Absence of such a correlation is indicative that partition measures surface properties other than charge at the plane of shear. Acetaldehyde- and glutaraldehyde-fixed erythrocytes were partitioned in two-polymer phases and their electrophoretic mobility examined in order to investigate the circumstances under which the above-indicated correlation holds. Aspects of the effect of fixation of cells on their surface properties was thereby obtained.

1. 1. Acetaldehyde-fixed and normal human red blood cells have similar partitions and similar electrophoretic mobilities; glutaraldehyde-fixed red cells display markedly increased partitions and mobilities.

2. 2. Lipid-extracted aldehyde-fixed cells have substantially increased partitions, but unchanged mobilities, when compared with the fixed cells from which they were prepared. This indicates that the removal of lipid exposes a higher density of charge groups deeper in the membrane than is measurable by electrophoresis under the conditions used.

3. 3. The countercurrent distribution obtained with fixed cells depends on the length of time chosen for phase settling. Short times result in a single distribution while longer settling times give rise to a two-peak distribution. The latter phenomenon probably arises from a time-dependent aggregation of the fixed cells in the phases.

4. 4. Electrophoretic examination of the glutaraldehyde-fixed cells from different cavities along the extraction train indicates that the fixation process does not eliminate differences in the relative electrophoretic mobilities of erythrocytes of different ages.

     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio (P) and the P itself, differences in membrane components suspected of effecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat red blood cells of different ages.     

Separation of cell mixtures by immunoaffinity cell partitioning: strategies for low abundance cells

The partitioning of cells in aqueous two-phase systems formed by poly(ethylene glycol) (PEG) and dextran can be changed by incubating the cells with a PEG-modified antibody directed specifically against its surface. We have developed a new approach for immunoaffinity cell partitioning (IACP) in which the antibodies are first reacted with tresylated monomethoxy PEG (TMPEG) in sodium phosphate buffer, pH 7.5, the excess TMPEG is quenched by reaction with bovine serum albumin, and the resulting preparation is used directly for incubation with the cells without any isolation of the monomethoxyPEG (MPEG)-antibody conjugates. We have demonstrated the specificity of this IACP method by showing that MPEG-modified anti-human red blood cell antibody increases the partition of human erythrocytes from the interface to the PEG-rich top phase (up to 100%) but not the partitioning of either neutrophils or HL60 cells. Irrelevant antibodies do not affect the partitioning of red blood cells. The partitioning behaviors of erythrocytes and HL60 cells in mixtures varying from 75 to 10% red blood cells subjected to IACP are similar to those of the pure cell population, i.e., erythrocytes ca. 100% and HL60 cells 3% in top phase. Thus, the population of erythrocytes can be almost completely extracted into the top phase in a single step. The contaminant cells represent only a small percentage (less than 5% in most of the cases) of the cell mixture recovered in top phase. Both cell populations can be completely separated by countercurrent distribution (CCD).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood clam (Anadara inflata) red cells. partition in aqueoues two-polymer phase systems

Anadara inflata is a clam which has red blood cells in its hemolymph. Furthermore, the nucleated red blood cells contain two structurally distinct hemoglobins. Clam red cells were subjected to partition in aqueous dextran-polyethylene glycol two-phase systems with the following results:

1.

1. Clam red cells are the largest cells (about 20 μm in diameter) so far studied in two-polymer phases. It is shown that not only can such cells be partitioned in dextran-polyethylene glycol phase systems, but that countercurrent distribution resolves the clam red cell population into more and less metabolically active cells. The distribution of these cells in relation to the whole population is similar to that of young and old red cells from mammals.     

Effect of rapidity of phase separation on the efficiency of cell fractionation by partitioning in aqueous two-phase systems

Partitioning in two-polymer aqueous phase systems is an established method for the separation, purification and characterization of biomaterials. Because of the relatively slow settling rates of these phases, a consequence of the slight difference in density between them, effort has been directed to speeding up phase separation by various means (e.g., the development of a thin-layer countercurrent distribution apparatus). This has resulted in the more rapid processing of materials. Unlike soluble materials, biological particulates (e.g., cells) generally partition between one of the bulk phases and the interface. The mechanism of cell partitioning involves cell-specific adsorption to droplets of one phase suspended in the other, subsequent to phase mixing, and the delivery of adsorbed cells to the bulk interface as the droplets settle. In this communication we show, using erythrocytes as a model, that speeding up phase separation is counterproductive when partitioning cells and results in reduced efficiency of their separation or subfractionation. The most likely reason for this result is that increasing the speed of phase settling removes the droplets of one phase suspended in the other more rapidly than cells can attach to them, thereby interfering with the mechanism whereby cells partition.     

The effect of neuraminidase on the relative surface charge-associated properties of rat red blood cells of different ages

Approx. 70% of the sialic acid on the rat erythrocyte surface is susceptible to cleavage by neuraminidase (Vibrio cholerae). Neuraminidase treatment results in a reduction in the partition coefficient (K) of the red cells in a charged dextran-poly(ethylene glycol) aqueous phase system and in the electrophoretic mobility of the cells. Countercurrent distribution of rat neuraminidase-treated erythrocytes, containing 59Fe-labeled mature red cells of distinct age, indicates that (a) the electrophoretic mobilities of red cells in different cavities along the extraction train increase with increasing K, as is the case with untreated erythrocytes, and (b) the cell age-related differences in surface charge-associated properties are neither eliminated nor altered by the enzyme action.     

Detection of Differences in Surface-charge-associated Properties of Cells by Partition in Two-polymer Aqueous Phase Systems

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems^1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means³. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning^4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh⁷, as opposed to the mean electrophoretic mobility of both cell populations which remain identical⁸.     

Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases

Human peripheral blood monocyte-enriched fractions (identified by staining for peroxidase and by sizing) were obtained by velocity sedimentation at unit gravity of peripheral blood mononuclear cells. They were then fractionated by countercurrent distribution (a multiple-extraction procedure) in a charged Dextran/poly(ethylene glycol) aqueous phase system. The monocytes remained viable after the separation (order of 90%). Cells obtained from different cavities along the extraction train were tested for their ability to phagocytize latex particles. With increasing partition coefficient (presumably higher charge-associated membrane properties) the ratio of monocytes that phagocytized to monocytes that did not phagocytize increased appreciably. When, however, monocytes were permitted to phagocytize particles prior to countercurrent distribution, an increase in partition coefficient was associated with an appreciable decrease in the above-specified ratio. Control experiments indicate that the observed change in partitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitiartitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitioning) and their ability to ingest particles. Furthermore, an alteration in the surface charge-associated properties of monocytes as a consequence of phagocytosis is indicated by the cells' reduced partition coefficient.     

Countercurrent distribution and multiple sedimentation of Chlorella pyrenoidosa during its life cycle

Synchronously and normally grown Chlorella pyrenoidosa cell populations were analysed by countercurrent distribution in aqueous two-polymer phase systems and by a multiple sedimentatation technique. Partition of cells in aqueous phases reflects the surface properties of cells (primarily surface charge) and multiple sedimentation reflects the cells' size-density parameters. It was found that:

1. 1. Synchronized cells that have just divided have the lowest partition of any in the population. Surface charge (as reflected by partition) increases with time after cell division. Cells have the highest partition just prior to division.

2. 2. Synchronized cells that have just divided are the smallest of any in the population. Since size and sedimentation rate increase with time after cell division multiple sedimentation permits the separation of cells of different ages.

3. 3. Both countercurrent distribution and multiple sedimentation studies reveal considerable heterogeneity of synchronized Chlorella populations. The increase in both surface charge and size with cell age does not appear to proceed in a continuous fashion. Rather, it seems to go in a stepwise manner.

4. 4. Non-synchronized cells examined by either countercurrent distribution or multiple sedimentation show two distinct sub-populations. One of these corresponds to the youngest, just divided cells; and the other to cells just prior to cell division. It is suggested that a lag time just prior to cell division and just after cell division explains these results.

5. 5. Countercurrent distribution in two-polymer phases and multiple sedimentation at unit gravity in a suspension medium best suited for the cell under investigation seem to be methods of choice for tracing cell changes during division, maturation and aging and for sub-fractionating such cell populations.