Age dependent phagocytosis of red cell membranes by the reticulohistiocytary system of the isolated perfused rat liver

Isolated rat livers were perfused with a medium free of immune globulins, protein and hemoglobin to investigate age-dependent differences in the phagocytosis of erythrocyte membranes. Membranes of young erythrocytes were phagocyted significantly more than those of old ones. The control parameters of the liver function also were significantly higher during the increased phagocytosis of the young cells than for the old cells and the control group. In the aged animals the characteristic of the elimination curve is changing. The old livers eliminated less ghosts than the young ones. These results are significant.     

The formation of heinz bodies in ghosts of human erythrocytes of adults and newborn infants

Summary Isolated membranes (ghosts) of human erythrocytes of newborn infants produce Heinz bodies faster after an exposure to acetylphenylhydrazine than the ghosts of adults. Heinz bodies form faster in ghosts prepared from erythrocytes of newborn infants, incubated in their own plasma or in the plasma of adults, than in ghosts of incubated red cells of adults. A direct relation can be seen between the hemoglobin content of the ghosts and the formation of Heinz bodies. It is suggested that the Heinz body susceptibility of normal human erythrocytes depends on the quantity of hemoglobin bound to the red cell membrane.This work was supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Grants No. Schr 86/8 and Schr 86/9.     

Age-dependent physiochemical and biochemical studies of human red cell membranes.

Erythrocyte ghosts of male persons of two age groups, younger than thirty and older than seventy years, were analyzed to investigate the imfluence of age on lipid composition, the physical state of the lipid phase, Na+K+-ATPase activity and sialic acid content. The phospholipid content in red cell membranes of old donors is significantly lower than in young ones. Cholesterol and fatty acid compositions shows no difference between the two donor groups. The membrane fluidity in liposomes prepared from total lipids of old donor decreased. No significant difference in lipid composition and membrane fluidity reflected by the spin labels was observed between blood group A and O. The activity of Na+K+-ATPase of the young donors with blood group A is significantly higher than those of old donors. The results also demonstrate a decrease of sialic acid content of red cells of old donors.     

The effects of pentoxifylline on rat erythrocytes of different age

Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug-induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca2+; (2) membrane protein phosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5-2.5 mM) reduced intracellular Ca2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0-5.0 mM) increased Ca2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inhibition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca2+ entry into the cell.     

Effect of in vitro carbamylation of erythrocytes on their rheological properties

In order to assess the role of carbamylation of erythrocyte proteins in the modification of rheological parameters of red blood cells observed in uremic patients, and in vitro carbamylation of erythrocytes and hemoglobin was carried out using sodium cyanate. The carbamylation of hemoglobin was determined by observation of the increase of HbA1 fraction. The deformability of erythrocytes and the viscosity of erythrocyte suspensions and of hemolysate were measured. The results showed an increase in the deformability of red blood cells and a decrease in the viscosity of hemoglobin as the carbamylation increased. This is attributed to a decrease of hemoglobin viscosity and to a modification of the electric charge of the membrane. These results show that the reduced erythrocyte deformability observed in patients with renal failure is not due to erythrocyte protein carbamylation.     

Hemolysis of human erythrocytes with saponin affects the membrane structure

Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin-lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.     

Anaphylactic histamine release and membrane fluidity of mast cells from young and aged rats

The effect of aging on mast cell secretory function was studied in young (8 weeks old) and aged (72 weeks old) female BD₆ rats using passive cutaneous anaphylaxis (PCA) and the histamine release testin vitro. Young rats were more susceptible to PCA and their peritoneal mast cells released more histamine in anin vitro anaphylactic reaction. Measurement of electron spin resonance (ESR) using methyl-5-doxylstearate and methyl-16-doxylstearate as spin labels demonstrated that cell membranes of mast cells of young rats showed higher fluidity in the resting state and a more pronounced increase in membrane fluidity after anaphylactic stimulation than mast cell membranes of aged rats. The study of lipid composition of mast cell membranes showed that the ratio of cholesterol (C) to phospholipids (P) was lower in mast cells of young rats than in mast cells of aged rats. This suggests that a decrease in membrane fluidity, associated with an increase in C/P ratio may be one of the factors responsible for decreased histamine releasability of mast cells from aged rats.     

Regulation of intracellular metabolism by biodegradable polyrotaxanes

Cellular response to our designed biodegradable polyrotaxanes was investigated in terms of changes in cytoplasmic calcium levels in platelets. The polyrotaxanes regulated thrombin-induced calcium increase in platelets although constituent molecules of the polyrotaxanes showed fewer effects on the intracellular metabolism. Further, an increase in membrane fluidity of red blood cell ghosts was significantly observed by the addition of the polyrotaxanes. Static light scattering study revealed that the polyrotaxanes formed a supramolecular association state in relation to the molecular weight of PEG: a loosely packed association with a specific molecular shape. From these characteristics, it is suggested that supramolecular level interactions between the polyrotaxanes and cell membranes regulate the intracellular metabolism. It is concluded that these biodegradable polyrotaxanes can be feasible as temporarily-controlled bioactivator.     

Acetylcholinesterase during aging of human erythrocytes

Acetylcholinesterase activity differs in the membranes of young, mature, and old human erythrocytes: It is highest in the mature and lowest in the old cells. The enzymes in young and mature erythrocytes is in the form of three, but in the old cells in the form of two molecular components. The results suggest that changes in the structural organization of acetylcholinesterase in the erythrocyte membrane have a direct bearing on the aging of red blood cells.Laboratory of Biochemistry, Institute of Hematology and Blood Transfusion, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. S. Il'in.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 10, pp. 1198–1200, October, 1976.     

Anaphylactic histamine release and membrane fluidity of mast cells from young and aged rats

The effect of aging on mast cell secretory function was studied in young (8 weeks old) and aged (72 weeks old) female BD₆ rats using passive cutaneous anaphylaxis (PCA) and the histamine release testin vitro. Young rats were more susceptible to PCA and their peritoneal mast cells released more histamine in anin vitro anaphylactic reaction. Measurement of electron spin resonance (ESR) using methyl-5-doxylstearate and methyl-16-doxylstearate as spin labels demonstrated that cell membranes of mast cells of young rats showed higher fluidity in the resting state and a more pronounced increase in membrane fluidity after anaphylactic stimulation than mast cell membranes of aged rats. The study of lipid composition of mast cell membranes showed that the ratio of cholesterol (C) to phospholipids (P) was lower in mast cells of young rats than in mast cells of aged rats. This suggests that a decrease in membrane fluidity, associated with an increase in C/P ratio may be one of the factors responsible for decreased histamine releasability of mast cells from aged rats.

     

低氧性肺动脉高压大鼠红细胞的变形性,膜流动性及外形变化

为探讨红细胞变形性变化在低氧性肺动脉高压形成中的作用及低氧时红细胞变形性变化机制，本文观察了低氧1、3、5周大鼠红细胞的变形性、膜流动性、红细胞形态、红细胞平均体积（MCV）及红细胞平均血红蛋白浓度（MCHC）变化。结果：（1）低氧1周红细胞变形性即明显下降（即红细胞滤过指数FI增高），低氧时间越长红细胞变形性下降越明显；且与肺动脉压、右心室压升高及右心室肥厚呈负相关。（2）低氧3周及5周大鼠异常形态红细胞数明显增多。（3）低氧5周大鼠红细胞膜流动性明显下降。提示：低氧大鼠红细胞形态异常及红细胞膜流动性下降可使红细胞变形性下降。红细胞变形性下降可能在低氧性肺动脉高压及右室肥厚的发生及发展中起一定的作用。     

Erythrocytes from young but not elderly donors can bind and degrade immune complex- and antibody-bound C3 in vitro.

In situ aged erythrocytes (senescent erythrocytes from young donors and both young and old erythrocytes from elderly donors) demonstrate high levels of membrane-bound C3c and C3d and elevated susceptibility to in vitro phagocytosis. In this study we demonstrate that in situ aged erythrocytes are defective in their ability to degrade C3 fragments and clear them from the erythrocyte membrane. Erythrocytes from young donors bind complement-bearing immune complexes via CR1 and become susceptible to complement-mediated erythrophagocytosis ('innocent bystander' sequestration). Erythrocytes from elderly donors are defective in their ability to bind such immune complexes, as attested by the lack of an increment in membrane-associated C3 fragments as detected by flow cytometry and lack of an increment in in vitro sequestration. Factor I (serum)-dependent cleavage of C3 fragments and release of immune complexes from the erythrocytes of young donors lead to a drop in erythrocyte-associated C3 fragments and the disappearance of the 'innocent bystander' phenomenon. Inhibition of Factor I, and thus inhibition of C3b degradation and immune complex release for the erythrocyte membrane, enhances the levels of 'innocent bystander' sequestration of erythrocytes from young donors. Erythrocytes from elderly donors are defective in the dynamic process of CR1 binding of complement-bearing immune complexes and Factor I-associated release of membrane-associated C3 fragments. A defect in the ability of all in situ aged erythrocytes to clear their membranes of C3 fragments is also demonstrated for complement bound to the erythrocyte via IgM isoagglutinins. While erythrocytes from both young and elderly donors allow for the IgM-mediated binding of complement to the erythrocyte, only young erythrocytes from young donors demonstrate degradative activity with the release of membrane-associated C3c. Erythrocytes from elderly donors demonstrate lower levels of detectable CR1 (CD35), decay accelerating factor (DAF) (CD55) and Protectin (CD59) than do erythrocytes from young donors. Time course studies determine that the defect in handling of immune complexes observed for in situ aged erythrocytes is not due to differences in the kinetics of loading or releasing of immune complexes. These findings on the refractiveness of erythrocytes of elderly donors to uptake of immune complexes and the degradation of C3 fragments may be of importance not only in the understanding of erythrocyte sequestration, but also in the physiology of immune clearance in the elderly.     

An experimental study of freezing in erythrocytes

A unique experimental apparatus (termed cryomicroscope) has been developed for the study of freezing and thawing processes in living cells and has been used specifically to determine the conditions requisite for intracellular freezing in human erythrocytes. Careful measurements of the incidence of intracellular ice formation have been correlated directly with the magnitude of the cooling rate during the freezing process and degree of extracellular supercooling prior to nucleation of ice. The probability for intracellular freezing increases with the magnitudes of both the cooling rate and the extracellular supercooling. A 3-dimensional contour has been defined experimentally by which the frozen state of human erythrocytes can be predicted as a function of these two parameters. The influence of cooling rate and extracellular supercooling on the mode of ice formation may be explained in terms of physiochemical phenomena which are controlled by the balance between the rates of heat transfer and mass transfer across the cell membrane during freezing.     

Effects of shape and size on red blood cell deformability: a static bending analysis

When flowing down a tapered tube, such as a narrow capillary, red blood cells (RBCs) are subject to deformation, the first event of which is folding in a pancake manner. The RBC deformability is reduced during cell ageing, a phenomenon that may reflect alterations in intracellular viscosity, membrane rigidity or RBC shape. Age related shape changes and their importance for increased RBC rigidity were theoretically analysed. The average empirically observed RBC profile is shown to offer little resistance to bending as compared to other, theoretically possible profiles of the same membrane area and RBC volume. Because of a decrease in projected area (diameter size), and therefore in pressure load, the pressure needed to initiate folding of an old RBC is between 20 and 55% higher than that required to fold a young one if, during RBC ageing, membrane area to cell volume ratio is constant as empirically observed. This difference exists whether the RBC is mathematically treated as a solid body or as a membrane shell.     

胰蛋白酶对红细胞膜动态力学特性的影响

目的： 研究胰蛋白酶对人红细胞膜表面电荷密度及动态力学特性的影响。 方法： 用密度梯度离心法从健康成人血液中分离出“青年”和“老年”红细胞，37 ℃下与不同浓度的胰蛋白酶孵育60 min。利用Zeta电位分析仪测量红细胞膜表面电荷密度，利用快速显微动态图像分析技术测定单个活态红细胞的大小、形态和细胞膜弯曲弹性模量（Kc）和剪切弹性模量（μc）。 结果： （1）正常和用胰蛋白酶处理后“老年”红细胞的表面电荷密度分别显著小于正常和用胰蛋白酶处理后的“青年”红细胞（P<0.01）。（2）正常“青年”红细胞和正常“老年”红细胞在形态上没有显著区别，但正常“老年”红细胞的接触面积显著小于正常“青年”红细胞（P<0.01）；胰蛋白酶处理可减小红细胞的大小，但对形状规化因子没有显著影响。（3）正常“老年”红细胞膜Kc和μc均显著大于正常“青年”红细胞（P<0.05）；“青年”和“老年”红细胞膜Kc和μc均随胰蛋白酶浓度的增加而增大。结论： 胰蛋白酶处理可降低“青年”和“老年”红细胞膜表面电荷密度，增加红细胞膜的弯曲弹性模量，从而降低红细胞膜的力学变形性。     

Effect of Membrane Cholesterol Depletion on the Immunoreactivity of the D Antigen and IgG Anti-D

The Immunoreactivity of the main Rh antigen (D) and Its corresponding antibody, as determined by a IgG to IgG combining ratio, antibody dissociation and antigen accessibility to antibody, was examined In cholesterol depleted human red cells and ghost membranes. The anti-IgG reactivity of IgG anti-D bound to cholesterol depleted red cells and ghosts was demonstrably enhanced in vitro and in electron microscopy studies, particularly in ghosts. Dissociation of cell bound anti-D during buffer incubation was greater after cholesterol depletion, especially in ghosts. There was also reduced binding of anti-D to cholesterol-depleted eel is as previously reported. All these effects appeared to be independent of endogenous or exogenous proteolysis in either cholesterol-depleted membranes or controls as Judged from membrane electrophoretic analyses. A₂C, an agent which increases membrane fluidity, had no effect on anti-D binding or the antiglobulin reactivity of cell bound IgG. A reduction in anti-D binding also was observed In red cells depleted of cholesterol following Immobilization of membrane proteins by glutaraldehyde crosslinking. The findings show that cholesterol depletion not only affects the antigen but also Rh antibody reactivity. They also suggest that factors other than vertical antigen movement in a fluid bi layer may influence the behavior of the D antigen in cholesterol-modified erythrocytes.     

红细胞老化过程中囊泡化作用的探讨

红细胞存活120天,在体内老化的过程中,不断产生囊泡,膜脂及膜蛋白随囊泡丢失,红细胞逐渐形成球形,易破溶而消亡。本文用密度梯度方法将年轻及老化红细胞分离,加Ca~(2+)及钙离子载体(A_(23187))诱导红细胞囊泡化,比较年轻及老化红细胞囊泡化后及释放出的囊泡膜蛋白的变化,观察囊泡化后红细胞的变形性、溶血度及被吞噬细胞吞噬的能力。实验结果发现在Ca~(2+)诱导下年轻红细胞比老化者更易囊泡化;释放的囊泡中主要含区带3蛋白及少量区带4.5及4.9,另外,区带7及Hb明显增多,囊泡化后的红细胞膜有聚集。囊泡化后的红细胞变形性明显降低,易溶血及被吞噬细胞吞噬的量明显增多。     

老化红细胞变形能力与膜钙依赖中性蛋白酶活性的关系

研究表明,老化红细胞变形能力明显降低,且其降低与血红蛋白浓度增高及膜弹性降低有关［１］。红细胞膜钙依赖中性蛋白酶（Ｃａｌｐａｉｎ）和它的内源性抑制剂（Ｃａｌｐａｓｔａｔｉｎ）形成红细胞中一个蛋白水解系统,参与红细胞中的信号传导,调节细胞形状、体积和细胞膜通透性,与高血压、细胞老化等生理、病理现象密切相关。Ｃａｌｐａｉｎ可限制性水解红细胞膜骨架蛋白和其它膜内蛋白,导致红细胞损伤［２,３］。而老化红细胞变形能力降低与Ｃａｌｐａｉｎ的关系尚不清楚,为此我们检测了４２例健康人老化及年轻红细胞变形能力、Ｃ…     

Spectrin phosphorylation in senescent rat erythrocytes

The rates of phosphorylation and dephosphorylation of the erythrocyte cytoskeletal protein, spectrin, were analyzed in young and old rat erythrocytes. Endogenous membrane protein kinase activity was measured in age-seperated rat erythrocytes, and was found to decrease as a function of cell age. Membranes prepared from young and old erythrocytes contained comparable levels of protein phosphatase activity. Spectrin phosphatase activity was readily observed in erythrocyte membranes.Partially purified spectrin kinase and spectrin were prepared from membranes obtained from young and old erythrocytes, and the phosphorylation of the spectrin fractions was measured with the isolated kinases. The kinases prepared from young or old cells phosphorylated spectrin from young cells to the same extent. When spectrin from old cells was used as the substrate, it was phosphorylated ten-fold less extensively by the spectrin kinase prepared from old cells than by the spectrin kinase from young cells. This finding indicated that the decrased phosphorylation of spectrin observed in membranes prepared from age-separated red cells was due to a structural alteration in the spectrin.A structural basis for the decreased phosphorylation of spectrin in older erythrocytes was sought. Treatment of erythrocyte membranes with malonyldialdehyde, a product of lipid peroxidation which accumulates in erythrocyte membranes during senescence, adversely affected spectrin phosphorylation. The results presented here indicate that intramolecular derivatization of spectrin was sufficient to impair its function as a substrate for protein kinase.     

Role of peroxidation in erythrocyte aging

The role of membrane peroxidation in red cell aging was investigated by exposing rat erythrocytes to a peroxide-generating system of xanthine oxidase with hypoxanthine, and the resulting alterations were compared to those observed during in vivo aging. Erythrocyte incubation with peroxides decreased the activities of adenylate cyclase and protein kinase, enzymes which were found to be reliable markers reflecting differences between young and old cells. Membranes from age-separated erythrocytes were solubilized and subjected to electrophoresis on gradient polyacrylamide-sodium dodecyl sulfate gels. Electrophoretic patterns indicated that old erythrocytes contained higher amounts of several membrane proteins than young cells. These were proteins in bands 2.1 and 4.1, and proteins of molecular weight larger than 250,000. There was a relative increase in the amount of proteins in band 4.1 and the high molecular weight (greater than 250,000) proteins as a results of peroxidation. These studies demonstrate that cells incubated in vitro with a peroxide-generating system resemble senescent erythrocytes in membrane enzyme activities, cell density, and membrane protein composition. We propose that peroxidation plays an important role in red cell aging.