Decreased Membrane Deformability Research Articles

Effect of Mercury on Membrane Proteins, Anionic Transport and Cell Morphology in Human Erythrocytes.

Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. For this aim, human RBC were treated in vitro with different HgCl2 concentrations (range 10-40 µM) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS- bearing MVs were quantified by annexin-V binding using FACS analysis. Findings presented in this paper indicate that RBC exposure to HgCl2 induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl2 treatment in a dose dependent manner. Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges.

Comparison of Hemoglobin Köln Erythrocyte Membranes With Malondialdehyde-Reacted Normal Erythrocyte Membranes

AbstractSplenectomized patients with hemoglobin (Hb) Köln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide-reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Köln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Köln RBC membranes, and MDA-reacted membranes with [3H]NaBH4. Hb Köln RBC membranes and MDA-reacted membranes both had significantly more 3H incorporation than control membranes. Furthermore, 3H incorporation in both Hb Köln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [14C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH4 prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH4 after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH4-reduced Hb Köln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Köln RBCs. We conclude that Hb Köln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [3H]NaBH4-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.

Sensing of red blood cells with decreased membrane deformability by the human spleen

AbstractThe current paradigm in the pathogenesis of several hemolytic red blood cell disorders is that reduced cellular deformability is a key determinant of splenic sequestration of affected red cells. Three distinct features regulate cellular deformability: membrane deformability, surface area-to-volume ratio (cell sphericity), and cytoplasmic viscosity. By perfusing normal human spleens ex vivo, we had previously showed that red cells with increased sphericity are rapidly sequestered by the spleen. Here, we assessed the retention kinetics of red cells with decreased membrane deformability but without marked shape changes. A controlled decrease in membrane deformability (increased membrane rigidity) was induced by treating normal red cells with increasing concentrations of diamide. Following perfusion, diamide-treated red blood cells (RBCs) were rapidly retained in the spleen with a mean clearance half-time of 5.9 minutes (range, 4.0-13.0). Splenic clearance correlated positively with increased membrane rigidity (r = 0.93; P < .0001). To determine to what extent this increased retention was related to mechanical blockade in the spleen, diamide-treated red cells were filtered through microsphere layers that mimic the mechanical sensing of red cells by the spleen. Diamide-treated red cells were retained in the microsphilters (median, 7.5%; range, 0%-38.6%), although to a lesser extent compared with the spleen (median, 44.1%; range, 7.3%-64.0%; P < .0001). Taken together, these results have implications for understanding the sensitivity of the human spleen to sequester red cells with altered cellular deformability due to various cellular alterations and for explaining clinical heterogeneity of RBC membrane disorders.

Characterizing pathology in erythrocytes using morphological and biophysical membrane properties: Relation to impaired hemorheology and cardiovascular function in rheumatoid arthritis

The inflammatory burden of the complex rheumatoid arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value <0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBC's capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell's morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function.

17th International Symposium of the European Association for Red Cell Research, EARCR 2009, Triuggio, Milano, Italy, April 23–27, 2009

The inflammatory burden of the complex rheumatoid arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value < 0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBC's capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell's morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function.

Modulation of Band 3-Ankyrin Interaction by Protein 4.1: FUNCTIONAL IMPLICATIONS IN REGULATION OF ERYTHROCYTE MEMBRANE MECHANICAL PROPERTIES

Protein 4.1 is an important structural component of the erythrocyte membrane. In contrast to our detailed understanding of the role of protein 4.1 in regulating membrane mechanical properties through modulation of spectrin-actin interaction, very little is known regarding the functional implications of protein 4.1 interaction with band 3. In the present study, we explored the potential role of protein 4.1-band 3 interaction in modulating membrane mechanical properties. Based on recent studies which identified the sequence motif IRRRY in band 3 as the protein 4.1 interacting domain, we studied the functional consequences of specific dissociation of band 3-protein 4.1 interaction by the synthetic peptide IRRRY. We show that protein 4.1 bound to the inside-out vesicles could be dissociated from band 3 but not from glycophorin C by IRRRY. Furthermore, incorporation of IRRRY into resealed ghosts resulted in decreased membrane deformability and increased membrane mechanical stability. The observed alterations in membrane properties appears to result from increased band 3-ankyrin interaction following dissociation of protein 4.1 from band 3. These studies have enabled us to identify an important functional role for band 3-protein 4.1 interaction in modulating erythrocyte membrane properties.

Membrane deformability and the capacity for shape change in the erythrocyte

Erythrocytes must have the capacity to undergo marked membrane deformation and shape changes in order to circulate through capillaries and respond to a range of shear stresses. To study the interrelationships between membrane deformability and the capacity for shape transformation, we created rigid membranes using several agents and then examined the ability of these erythrocytes with rigid membranes to undergo amphipath-induced shape change. We have previously shown that wheat germ agglutinin (WGA) and a monoclonal antibody to glycophorin A (R-10) cause membrane rigidity as measured by ektacytometry. Experiments were therefore designed to produced comparably rigid membranes using WGA, R-10, and diamide, and then to test the ability of lysophosphatidylcholine to produce echinocytes, and primaquine to produce stomatocytes. We found that diamide treatment substantially blocked both types of shape change. In contrast, R-10 binding did not impair either primaquine- or lysophosphatidylcholine- induce shape change. Further, WGA blocked echinocyte transformation, as previously reported, but not stomatocytosis. Using reduced and unreduced gel electrophoresis and Triton extraction, we compared the biochemical changes associated with WGA-, diamide-, and R-10-induced rigidity, and found them to be different. We conclude that not all rigid cells are incapable of shape change, and therefore that decreased membrane deformability is not predictive of impaired capacity for shape change.

The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes

The formation of spectrin-hemoglobin complex following treatment of red cells with hydrogen peroxide (H 2O 2) has previously been shown to be associated with alterations in cell shape, decreased membrane deformability and increased recognition of modified cells by anti-IgM immunoglobulin in a phagocytic assay by monocytes. Prior treatment with carbon monoxide completely inhibited the H 2O 2-associated membrane changes, indicating a role for oxidized hemoglobin in the complex formation. Also, in a cell-free system, blockage of sulfhydryl (SH) groups on purified spectrin by N- ethylmaleimide significantly reduced the complex formation, suggesting a role for SH groups of spectrin in crosslinking process. The present study was undertaken to examine the role of SH blockade by N- ethylmaleimide on intact red cells undergoing oxidative damage. Pretreatment of erythrocytes with N- ethylmaleimide at concentrations ranging from 0.1 to 0.2 mM resulted in decreased lipid peroxidation and spectrin hemoglobin crosslinking. Moreover, pretreatment with N- ethylmaleimide resulted in less marked alterations in cell shape and membrane deformability as well as reduced recognition of peroxidized cells by antiglobulin serum. N- Ethylmaleimide treatment had no effect on methemoglobin formation. Studies with 14C-labeled N- ethylmaleimide showed that over 50% of N- ethylmaleimide was incorporated into spectrin. Pretreatment of cells with higher concentrations of N- ethylmaleimide (over 0.2 mM) was associated with membrane dysfunction independent of H 2O 2. These results imply that blocking of reactive SH groups leads to reduced interaction of spectrin with oxidized globin. These data, along with our prior observations, indicate that SH groups on spectrin play an important role in hemoglobin oxidation-induced formation of spectrin-hemoglobin complex and the resultant deleterious effects on membrane properties.

Effect of complement on the viscoelastic properties of human erythrocyte membrane.

Using the micropipette technique, we examined the viscoelastic properties of the red blood cell (RBC) membrane which had been strongly coated with various components of complement (primary C3b, C3d, C4b, C4d) in vitro. The membrane elastic modulus (E), the viscosity index of the initial rapid phase of deformation (eta D1), the viscosity of the later slow phase of deformation (eta D2) and the viscosity of the recovery phase (eta R) were determined. Compared to control non-complement coated RBCs, RBCs coated with C3d, either alone or with other complement components, showed significant increase in the values for elasticity and viscosities. Thus C3d fixation resulted in decreased membrane deformability. Changes in membrane viscoelasticity due to bound C3d were not enhanced by bound C4b, C4d, C5, factor Bb or p; presence on RBC membrane of the latter two complement components may partially reverse the effect of C3d fixation. Lipid fluidity of RBC membrane, examined by fluorescence depolarization, increased with fixation of all complement components except C5. These complement-induced changes in membrane viscoelastic properties have potential pathophysiological and clinical implications. The data suggest that extravascular sequestration of human RBCs may be explained in part by increased membrane rigidity resulting from C3d fixation.

Decreased membrane deformability in Melanesian ovalocytes from Papua New Guinea.

We examined the ability of Melanesian ovalocytes from Papua New Guinea to be deformed in order to probe the resistance of these cells to invasion by several species of malaria parasite. We found ovalocytes were refractile to drug-induced endocytosis, that they formed abnormal rouleaux, showed reduced deformability when aspirated into 0.6-micron diameter pores in polycarbonate sieves, and failed to crenate when mounted under a glass coverslip. No substantial differences were found between normocytes and ovalocytes in their initial rate of filtration through 4.5-micron pore polycarbonate sieves, their membrane fluidity as measured by the rate of depolarization of fluorescent probes or the rate of extraction of cytoskeletal proteins in low ionic strength buffers. We conclude that the resistance of ovalocytes to undergo localized deformation might be significant in explaining the resistance of these cells to invasion by malarial merozoites.

Oxidant damage of the lipids and proteins of the erythrocyte membranes in unstable hemoglobin disease. Evidence for the role of lipid peroxidation.

Since unstable hemoglobins have been considered a source of reactive oxygen radicals, and oxidative membrane damage a prehemolytic event, we examined the erythrocyte membranes of six patients (three splenectomized) with hemoglobin Köln disease. In the hydrogen peroxide stress test, the patients' erythrocytes generated more than twice the malonyldialdehyde (a lipid peroxidative product) than control erythrocytes. Fluorescence spectra of lipid extracts of the patients' erythrocytes showed an excitation maximum at 400 nm and an emission maximum of 460 nm, characteristic of malonyldialdehyde lipid adducts. Two types of membrane polypeptide aggregates were found in the erythrocytes of the splenectomized patients. The first, which were dissociable by treatment with mercaptoethanol, contained disulfide-linked spectrin, band 3 and globin. The second, not dissociable by mercaptoethanol, had an amino acid composition similar to that of erythrocyte membranes and spectrin (unlike globin) and like that of aggregates produced by the action of malonyldialdehyde on normal erythrocyte membranes. Atomic absorption spectroscopy of hemoglobin Köln erythrocytes showed no increase in calcium content implying that these cross-links were not due to calcium-stimulated transglutaminase. Using a micropipette technique, we demonstrated that erythrocytes containing membrane aggregates from splenectomized patients were less deformable while aggregate-free erythrocytes from non-splenectomized patients had normal deformability. We conclude that the erythrocyte membranes in hemoglobin Köln disease show evidence of lipid peroxidation with production of malonyldialdehyde, and that the nondissociable membrane aggregates formed in this disease are likely cross-linked by malonyldialdehyde. Because the erythrocytes containing membrane aggregates from splenectomized patients with unstable hemoglobin disease show decreased membrane deformability, we hypothesize that this abnormality results in premature erythrocyte destruction in vivo.

Stabilization of erythrocyte membranes by polyamines.

Using a laser diffraction technique, we have studied the effects of putrescine, spermidine, and spermine, the three physiologic polyamines, on the deformability and mechanical stability of human erythrocyte membranes. Ghosts resealed with polyamines were subjected to high fluid shear stress in an ektacytometer. All polyamines increased the membrane shear modulus (decreased deformability) in a concentration- and time-dependent manner. The order of effectiveness was spermine greater than spermidine greater than putrescine. At 10 microM, spermine appreciably decreased membrane deformability. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytometer and deformability was continuously recorded as the deformable ghosts fragmented into rigid spherical vesicles. Polyamines, especially spermine, caused a noticeable increase in the t1/2 for fragmentation. These effects could not be ascribed to proteolysis or Ca2+-induced transglutamination. That the effects of polyamines were specific and not simply due to their positive charge was demonstrated by the finding that Ca2+ and Mg2+ destabilized the erythrocyte membrane as evidenced by decreasing the t1/2 for fragmentation. Extracellular polyamines were not effective except under conditions that caused significant accumulation inside the cell. The data indicate that intracellular physiologic polyamines, especially spermine, decrease erythrocyte membrane deformability and stabilize the membrane skeleton, making it more resistant to fragmentation.

Cell elastimetry in the detection of antineutrophil antibodies

Cell elastimetry has been applied to the measurement of antineutrophil antibodies. This technique measures, under direct visualization, the negative pressure required of aspirate PMNs into small-pored pipettes. Two groups of studies were carried out: (A) In the first group of studies, normal PMNs were incubated with 1 of 8 known antineutrophil serums. Each serum significantly decreased membrane deformability-- i.e., cells became more rigid. The study was conducted in an entirely blind fashion. Randomly coded serums from patients and controls were studied for deformability by observers unaware of the code. (B) In the second group of studies, sera containing immune complexes were incubated with normal PMNs. No significant effects were noted upon deformability. As a single cell assay that partially reflects membrane rigidity, elastimetry may, therefore, have potential in the further characterization of mechanisms by which such antineutrophil antibodies compromise neutrophil functions.

Cell Elastimetry in the Detection of Antineutrophil Antibodies

Cell elastimetry has been applied to the measurement of antineutrophil antibodies. This technique measures, under direct visualization, the negative pressure required to aspirate PMNs into small-pored pipettes. Two groups of studies were carried out: (A) In the first group of studies, normal PMNs were incubated with 1 of 8 known antineutrophil serums. Each serum significantly decreased membrane deformability—i.e., cells became more rigid. The study was conducted in an entirely blind fashion. Randomly coded serums from patients and controls were studied for deformability by observers unaware of the code. (B) In the second group of studies, sera containing immune complexes were incubated with normal PMNs. No significant effects were noted upon deformability. As a single cell assay that partially reflects membrane rigidity, elastimetry may, therefore, have potential in the further characterization of mechanisms by which such antineutrophil antibodies compromise neutrophil functions.

Analysis of factors regulating erythrocyte deformability.

Using a laser diffraction technique, we have studied factors that influence the deformability of erythrocytes. Variations in suspending medium osmolality and applied shear stress were employed to isolate the individual contributions to whole cell deformability of internal viscosity, surface area-to-volume ratio, and viscoelastic properties of the membrane. An experimental system was devised in which normal cells were modified in vitro to induce specific alterations in each factor. Measurements of deformability as a function of medium osmolality showed characteristic behavior of the modified cells. Reduced surface area-to-volume ratio was detected by an exaggeration of the normal decrease in deformability as medium osmolality was decreased. In contrast, increased internal viscosity was detected by an increase in deformability as osmolality was decreased. Finally, decreased membrane flexibility was detected by reduced deformation at low shear stress. These methods of analysis were applied to cells from patients with hereditary spherocytosis, hereditary pyropoikilocytosis, and hemoglobin CC disease to define the basis of reduced deformability. Hereditary spherocytes showed the combined effects of reduced surface area and increased internal viscosity. Hereditary pyropoikilocytes revealed the effects of severely reduced surface area-to-volume ratio. Hemoglobin CC cells showed only the effects of high internal viscosity. An increase in the membrane shear modulus (decreased membrane deformability) was not evident in these disorders.

Effect of anti-spectrin antibody and ATP on deformability of resealed erythrocyte membranes.

Deformability of resealed erythrocyte membranes was measured by using an ektacytometer. Divalent anti-spectrin antibody, but not monovalent anti-spectrin Fab fragments, decreased membrane deformability. Membranes resealed with MgATP were more deformable than those without MgATP. Exogenous alkaline phosphatase, which dephosphorylates spectrin, decreased membrane deformability. These results suggest that spectrin is an essential component of the system that determines erythrocyte deformability. They are consistent with the view that the role of ATP in membrane deformability is mediated through phosphorylation of the spectrin.

CELL ELASTIMETRY IN THE DETECTION OF IMMUNE NEUTROPENIA - - DEMONSTRATION OF A MEMBRANE PERTURBATION

Anti-neutrophil antibodies (ANA) have been demonstrated in the serums of recipients of multiple transfusions, mothers of infants with transient neonatal neutropenia, and in some patients with idiopathic neutropenia. Ingestion of sensitized neutrophils by other phagocytic cells has been observed, but direct evidence of membrane alteration of polymorphonuclear leukocytes (PMNS) by ANA is limited. We have used cell elastimetry to explore the problem. This technique measures, under direct visualization, the negative pressure required to aspirate PMNS into small pored pipettes. We have previously shown that the assay primarily reflects rigidity or deformability of the cell membrane. When incubated with normal PMNS, eight of nine serums from patients with known ANA significantly decreased membrane deformability -- i.e. cells became more rigid. The mean negative pressure required to aspirate PMNS incubated in normal plasma was 11.4 ± 3.6 (2 S.D.) cm Hg. PMNS incubated in the various ANA positive serums ranged from 25.9 to >43 cm Hg negative pressure required for aspiration. To insure objectivity, the study was conducted in an entirely blind fashion. Randomly coded serums from patients and controls were studied for deformability by observers unaware of the code. We conclude: 1) Elastimetry is a reliable and sensitive probe for the detection of ANA; 2) A primary effect of ANA upon PMN membranes has been demonstrated.

DECREASED MEMBRANE DEPORMABILIIY OF NEONATAL LEUKOCYTES AND ITS RELATIONSHIP TO NEUTROPHIL MOVEMENT

In previous studies we have demonstrated two separate mechanisms by which human neutrophils (PMNS) move:1)chemotaxis, or directed migration, and;2)random, or non-directed migration. We have also shown that neonatal PMNS are deficient in chemotaxis but have normal random migration. In order to further characterize the nature of neonatal PMN movement, we have employed the study of membrane deformability. Using a micromanipulator, individual PMNS were directed to the orifice of 3-5u internal diameter hollow glass pipettes. The “deformability” of the membrane was measured by the amount of negative pressure required to draw the cell into the pipette. The assay is precise and highly reproducible. Neonatal PMNS showed markedly decreased membrane deformability over control PMNS-i.e., were much more rigid cells. The finding of a functional abnormality of the neonatal PMN membrane is unique and has major significance:1)Since neonatal PMNS are deficient in chemotaxis but normal in random mobility, membrane deformability may play a role in chemotaxis. This is supported by the observation that iodoacetate, NaF and deoxyglucose, agents which decrease membrane deformability also selectively inhibit chemotaxis;2)PMN membrane deformability is an active process. The deformability defect here may, therefore, reflect metabolic immaturity of the neonatal PMN.