British Journal of Haernatology, 1981,47,38>350.

Microvesicles from Sickle Erythrocytes and their Relation to Irreversible Sickling

DAVID ALLAN, ANTHONY R. LIMBRICK,* PAUL THOMAS AND MAXWELL P. WESTERMANt

Department of Experimental Pathology, School of Medicine, and *Department of Biophysics, University College London

(Received 21 December 1979; accepted for publication 8 September 1980)

SUMMARY. Incubation of sickk (HbS) erythrocytes for periods up to 96 h leads to the formation of irreversibly sickled cells (ISCs) and to the release of spectrin-free microvesicles similar to those derived from aged or Ca’+-ionophore-treated normal erythrocytes. The sickle microvesicles were somewhat larger than those from normal cells and showed minor differences in their membrane polypeptide composi- tion. Sickle microvesicles were no different from their parent cells in their content of fetal haemoglobin. Neither microvesiculation nor formation of irreversibly sickled cells required the presence of Ca2+ in the medium but Ca2+ did accelerate both processes. Although in these prolonged incubations microvesiculation appeared to occur concomitantly with the formation of ISCs, it is not clear whether or not microvesiculation is a necessary prelude to irreversible sickling.

Sickle cell disease is a hereditary defect of the red blood cells and is characterized by the production of an abnormal haemoglobin (HbS) which under conditions of low oxygen tension undergoes a paracrystallization to produce the typical abnormally-shaped sickled erythrocyte. Under some circumstances, the cells may become impossible to restore to their normal discocyte form following reoxygenation even though their haemoglobin reverts to its normal soluble state, so it appears that these cells (irreversibly sickled cells, ISCs) have suffered an irreversible alteration to their membranes. Such cells are much less deformable than normal erythrocytes and may contribute to the initiation of microvascular occlusive events common in sickle cell disease. Thus although the primary biochemical lesion resides in the faulty amino-acid sequence of HbS, most of the clinical problems may result from a secondary defect in the cell membrane which prevents the restoration of normal cell morphology (Palek, 1977; Eaton et al, 1979).

The nature of the membrane lesion in ISCs has been a subject ofinvestigation for many years and a number of different factors have been suggested to influence the formation of ISCs. Among these are changes in Ca2+ (Eaton et al, 1973,1978; Palek, 1973), K+ and water contents (Glader et al, 1978; Glader & Sullivan, 1979; Clark et al, 1980), possible decreases in ATP

t Present address: Department of Medicine, Mount Sinai Hospital Medical Center, 15th St and California Avenue, Chicago, Ill. 60608, U.S.A.

Correspondence: Dr David Allan, Department of Experimental Pathology, School of Medicine, University College London, University Street, London WClE 6JJ.

0007-1048/81/0300-0383$02.00 0 1981 Blackwell Scientific Publications

383

384 David Allan et a1

concentrations (Glader et 01, 1978; Jensen et al , 1973; Lessin et al., 1978), alterations in the spectrin-actin framework (Lux et a/ , 1976; Lux, 1979), membrane protein cross-linking (Lorand et al, 1979) and loss of membrane components by fragmentation (Padilla et al , 1973). Other changes include decreased fetal haemoglobin concentration (Bertles & Milner, 1978) which might occur by selective loss during cell fragmentation (Yo0 et al, 1975) and increased haemoglobin binding to the cell membrane (Lessin et al, 1978).

Previous studies have examined the microvesiculation process which occurs in normal erythrocytes during ageing in vitro (Rumsby et al, 1977; Lutz et al, 1977; Shukla et al , 1978) or following treatment with Ca'+ ionophore A23187 (Allan ef al, 1976, 1980; Shukla et a l , 1978) but the loss of membrane components from sickle erythrocytes has only been partially characterized (see Westerman et al, 1979). In the present work we have shown that sickle erythrocytes lose membrane in a manner analogous to normal cells undergoing the above treatments and that during prolonged incubations in vitro a t 37°C this microvesiculation of sickle cells appears to be concomitant with the process of irreversible sickling.

METHODS

Seventeen patients were studied, all of whom had homozygous sickle cell anaemia. The PCV range was 2&30%, reticulocyte range was 8-27%, fetal haemoglobin range was 2.5-13% and endogenous level of ISCs was 2-8%.

Blood was collected in heparin and the red cells were separated by centrifugation and washed three times with 0.9% saline. Cells were incubated a t 37°C and 10% PCV in Krebs-Ringer solutions in which the normal bicarbonate buffer was replaced by 20 mM Hepes-NaOH buffer and which contained cithcr 1 mM CaC12 or 1 mM EGTA. All solutions contained penicillin (200 u/ml) and streptomycin sulphate (100 pg/ml). In some cases glucose (10 mg/ml) was added. Control samples of normal erythrocytes were incubated in parallel with the sickle cells.

Incubations of 10 ml aliquots were conducted in either 100% N2 or in room air for various periods in a shaking water bath (100 oscillations per min). N2 overlaying was obtained by allowing specimens to equilibrate for 45 min in a sealed glove box (Gallenkamp) which had been flushed with 100% N1. Residual oxygen tension in the sealed box was less than 1 mmHg. The percentage of irreversibly sickled cells was determined by counting 1000 cells after oxygenation in room air for 30 min and fixation in buffered saline (130 mM C1,20 mM sodium phosphate, pH 7.4) containing 2 % glutaraldehyde. Cells whose length was greater than twice the width and which possessed one or more pointed extremities under oxygenated conditions were considered to be irreversibly sickled (see Lessin et al, 1978).

After various periods of incubation, cells were sedimented a t 500g for 5 min and microvesi- cles (ifpresent) were isolated from the supernatant solution by centrifugation at 15 000g for 15 min. The microvesicles formed a firm bright-red pellet sometimes overlain by a pink, flocculent pellet of ghosts (in those cases where lysis was evident) which was removed by aspiration. Quantitation of microvesicles w a s achieved by resuspension of the red pellet in 1 ml O f 0-5°/o Triton XlOO followed by measurement of the optical density of the clear solution at 550 nm. It was established in two experiments that optical density measurements at 550 nm gave results that were relatively the same as measurements of phospholipid and cholesterol

Microvesiclesfrorn Sickled Erythrocytes 385

content in the microvesicles. Cell lysis was determined by measurement of the optical density at 550 nm of the clear supernatant solution remaining after sedimentation of the microvesicles.

Larger samples of microvesicles for biochemical and morphological analysis were prepared from both sickle and normal cells following incubation of up to 100 ml of cell suspension at 37°C for 24 h in the absence or presence of Ca2+. Ghosts were prepared from sickle cells after various periods of incubation. The cells were lysed and the ghosts washed in 10 mM Tris HCl buffer, pH 7.5, containing 0.2 mM EGTA. Methods for lipid analysis and electron microscopy were as recently described (Allan et al , 1980). Polypeptide analysis by SDS gel electrophoresis was performed on polyacrylamide slab gels by the procedure of Laemmli (1970). Samples containing about 20 pg of membrane protein or 200 pg ofmicrovesicle protein were applied to the gels. Haemoglobin types were characterized by starch gel electrophoresis at pH 8.4 (Huehns, 1968) and by the alkaline denaturation method of Singer et al (1951).

RESULTS

Fig 1 illustrates the characteristic morphology of irreversibly-sickled cells following incuba- tion for 24 h in the presence of EGTA. Very similar results were obtained with incubations in the presence of Ca’+.

Fig 2 shows a typical experiment in which the time courses of irreversible sickling and microvesicle release were compared in the presence or absence of Ca2+. Incubation was in a N2 atmosphere and no glucose was present in the medium. Maximum release of microvesicles and

incubation period ( h ) incubation period ( h 1

FIG 2. Time course of (a) irreversible sickling ofHbS erythrocytes (squares), (b) release ofmicrovesi- cles (sedimentable haemoglobin) (circles) and cell lysis (soluble haemoglobin) (triangles). In (b) values for release of microvesicles from normal erythrocytes are represented by squares. Filled symbols refer to incubation in the presence of Ca2+ and open symbols to incubation in medium containing EGTA instead of Ca2+. In (a) the ordinate values are presented as percentage oftotal cells which are irreversibly sickled. In (b) values for microvesicle release are expressed as percentages of the maximum observed release. Values for cell lysis are percentages of total cell haemoglobin found in the supernatant solution after sedimentation of cells and microvesicles.

386 David Allati et a1

loor 8

0 0 0

I 0 0 0

0

75t 0 0

0 0

0

0

50

0 . 0

25 1 0 0 0 0

0 . I I I I

0 25 50 75 100 % maximum release of microvesicles

FIG 3. The relationship between irreversible sickling and release ofmicrovesicles from cells incubated under N l in the absence of glucose. Results represent thc pooled data from five separate experiments similar to that shown in Fig I . Open circles refer to incubation in the absence of&'+, filled circles to incubations in the presence ofCa'+.

the maximum level of irreversible sickling were both achieved by 24-48 h in the presence of Ca?+. Both parameters increased a t a slower rate in the absence of Ca2+ (i.e. with EGTA substituted for Ca'+), but by 96 h, levels of microvesiculation and irreversible sickling approached those seen at earlier times in the presence of Ca2+. Cell lysis was higher in the presence of Ca'+ but generally did not exceed 10% after 96 h incubation. The maximum extent of microvesicle release corresponded to about 9% of the total cell phospholipid or about I"/" of the total haemoglobin. For reasons that are not understood, the yields of microvesicles usually diminished a t 96 h incubation compared with 48 h, especially in the presence of Ca2+. In experiments where incubations were carried out either in air or in the presence of glucose, microvesiculation and irreversible sickling were greatly diminished: microvesiculation levels were less than 10% of those seen with incubation under N2 and in the absence of glucose, and irreversible sickling remained at the levels present in the original blood samples. Normal cells incubated under N? and without glucose showed much less vesiculation, but these cells (especially in the presence of Ca'+) suffered higher levels of lysis than did sickle cells. Thus it was not possible to obtain values for vesiculation of normal cells later than 48 h. In Fig 3, the data from several experiments (each with blood from different donors) is combined to show the relationship between the degree of irreversible sickling and the extent of microvesicle release at each time point. The correlation between the two parameters is excellent in the presence of Ca'+ (correlation coefficient 0.76) but less good in its absence (correlation coefficient 0.71).

Electron microscopy of the microvesicles (Fig 4) demonstrated that they were rather similar in morphology to those derived from normal cells (Rumsby et al, 1977; Lutz et a l , 1977; Shukla

Microvesicles fuom Sickled Erythrocytes

Frc; 1. Light micrograph of sickle erythrocytes after 24 h incubation under N: in EGTA medium (see Methods section). Nomarksi optics, magnification x 1750. This micrograph waT kindly prepared by Dr Raoul Fresco.

FIG 4. Electron micrograph of microvesicles from sickle cells incubated for 24 h in a medium containing EGTA. x 30 000.

(Facing p . 386).

Frc. 3. SDS polyacrylamide gels of membranes and microvesicles from normal and sickle cells incubated for 24 h under N2. (a) Membranes from normal cells incubated in the presence ofEGTA. (b) Membrane5 froni normal cells incubated in the presence ofCa2+. (c) Membranes from sickle cells incubated in the presencc o f EGTA. (d) Membranct from tickle cells incubated in the prescnce of Ca”. (e) Microvesicle$ from normal cclls incubated in the presence of EGTA. (f) Microvesicles from sickle cells incubated in the presence of EGTA. (g) Microvesicles from sickle cclls incubated in the pretence of Ca’+. (h) Cytopla5m from normal c.clls. (i) Cytoplasm from sickle cells. Bands are numbered according to the nomenclature of Steck (197.1)

Microvesicles from Sickled Erythrocytes 387

et al , 1978), consisting predominantly of small membrane-bounded spheres containing dense material assumed to be erythrocyte cytoplasm. Although some ofthese spheres were similar in size to those from normal cells (about 150 nm), there were also many larger spheres ranging in size up to about 400 nm. Small numbers of ‘sausage’ or ‘dumbell’-shaped structures (myelin forms) similar to those in microvesicle preparations from ATP-depleted cells (Lutz, 1978) were also observed; these extended structures may represent intermediate forms between budding processes extending from the surface of cells and the fully separated microspheres making up the bulk of the preparation. No evidence was seen for the presence of membraneous appen- dages to the sickle microvesicles (‘tails’) similar to those observed on microvesicles from normal cells (Allan et al, 1976; Shukla et al , 1978). Microvesicles from sickle cells incubated with or without Ca2+ showed no apparent differences.

Fig 5 shows the polypeptide patterns of ghosts and microvesicles from normal and sickle erythrocytes. Few differences were seen between ghosts from normal and from sickle cells incubated for 24 h under N2. However, sickle ghosts generally were more difficult to prepare free of cytoplasmic contaminants and their electrophoretic patterns (Figs 5c, d) showed increased amounts of these components, e.g. haemoglobin, and bands in the region of 4.3 and 7 which also appeared in gels of erythrocyte cytoplasm (Fig 5h, i). An increased amount of band 4.3 was often but not always seen in ghosts from normal cells incubated for 24 h (Fig 5b). Microvesicles from sickle cells (Fig 5f, g) resembled those from normal cells (Fig 5e, and see also Rumsby et al , 1977; Lutz et al, 1977; Shukla et al , 1978); the spectrin bands (1,2) were also largely absent and the major remaining membrane protein was band 3. Other protein components of the microvesicles appeared to be derived largely from erythrocyte cytoplasm. No significant differences were seen between the polypeptide patterns of sickle microvesicles obtained by incubation of cells in the presence of Ca2+ and those obtained in the absence of Ca2+. Neither were variations in polypeptide pattern observed in microvesicles or ghosts prepared from cells incubated for different periods between 0 and 48 h.

TABLE I. Composition of microvesicles derived from sickle erythrocytes

+ Ca’+ +EGTA

(pmol/mg) 0.263 f 0.032 (4) 0.254f 0.027 (4) Phospholipid

Protein Cholesterol

Phospholipid

Phospholipid composition (mol %)

(molar ratio) 057+0.05 (3) 0.59+0.04 (3)

Sphingomyelin 26f2 (3) 26f1 (3) Phosphatidylcholine 30f1 (3) 29f2 (3) Phosphatidylserine 14k2 (3) 14f1 (3) Phosphatidylethanolamine 31 k 2 (3) 30+ 1 (3) Phosphatidate 1.5 (2) 1.0 (2)

Cells were incubated under Nr without glucose for 24 h and microvesicles were isolated as described in Methods. Incubations contained either 1 miv Ca2+ or 1 mM EGTA.

388 David Allan et a1

The lipid and protein composition of the sickle microvesicles was similar to the composi- tions previoudy reported for microvesicles from normal red cells (Rumsby et al , 1977; Lutz et al , 1977; Shukla et al , 1978) (Table I ) . Protein:lipid ratios for the sickle microvesicles were higher than the corresponding values for 'normal' microvesicles, consistent with the larger average size of the former. Cholesterol: phospholipid molar ratios were similar to values previously reported for whole sickle cells (Westerman et a\, 1979) and in the present experi- ments did not differ markedly from the ratio seen in membranes isolated from sickle cells (data not shown). The content of individual phospholipid classes in the sickle microvesicles was indistinguishable from values reported for whole sickle or normal erythrocytes (Westerman et al, 1963, 1979). In no case was a significant difference in lipid composition observed between sickle microvesicles prepared in the presence or absence of Ca2+.

DISCUSSION

The work presented here confirms previous indications of membrane loss from sickle erythro- cytes and characterizes more precisely than hitherto the nature of the microvesiculation process which is responsible for the loss of membrane. The small size of the microvesicles accounts for the apparent paradox that approaching 10% of the cell phospholipid may be lost while only about 1 % of the haemoglobin leaves the cells. I t is clear that microvesiculation of sickle cells is a process closely comparable to microvesiculation of normal cells, either aged, ATP-depleted or treated with Ca'+ ionophore A23187 (Allan et al, 1976; Rumsby et al , 1977; Lutz et a l , 1977; Shukla et a l , 1978; Lutz, 1978). Apart from the repeated observation that sickle microvesicles were on average somewhat larger (Fig 4) than those from normal cells the differences in structure and composition were rather small (Table I, Fig 5 and Allan et at, 1976; Rumsby et al, 1977; Lutz et a l , 1977; Shukla et al, 1978; Lutz, 1978). The phospho1ipid:protein ratio for the sickle microvesicles was only about 50-70% ofthat for microvesicles from normal cells (Lutz et al , 1977; Shukla et al , 1978), consistent with the larger average size of the sickle microvesicles.

Lutz et a1 (1977) found an unusually high content of phosphatidic acid in microvesicles from aged normal cells but we have not been able to confirm this observation in sickle microvesicles or those produced with A23187 (Table I and Allan et al , 1976). We have observed an increased content of the closely related lipid 1 ,Zdiacylglycerol in microvesicles from normal cells (Rumsby et a l , 1977; Allan et al, 1976) and have proposed that 1,2-diacylglycerol and phosphatidate production may be involved in the mechanism of microvesiculation (Allan et al, 1978). However, insufficient material was available in the present work to obtain reliable values for the diacylglycerol content of sickle microvesicles.

The time course over which sickle cells lose microvesicles is similar to that for aged normal cells (Lutz et a l , 1977) and considerably slower than the release of microvesicles from normal cells exposed to Ca'+ and ionophore A23187 (Allan et al, 1976, 1980). Release of microvesicles from sickle cells, however, was markedly inhibited by aeration of the cells or by addition of glucose to the medium. Both of these treatments also inhibit the formation of irreversibly sickled cells (ISCs) so that these results provided an indication of a possible link between ISC formation and microvesiculation. Such a link is confirmed on a temporal basis by the results in Fig 2. In five separate experiments where sickle cells were incubated with or without Ca2+ and where microvesiculation and ISC formation were measured (Fig 3), there appeared to be a

Microvesiclesfvom Sickled Erythrocytes 389

fairly good correlation between these two parameters, especially in those incubations where Ca2+ was included. It is not possible to decide from these results whether (a) microvesiculation precedes and promotes ISC formation, (b) ISC formation precedes and promotes microvesicu- lation, or (c) some other process is occurring which stimulates ISC formation and microvesicu- lation simultaneously.

Other workers have noted that ISCs often have a raised cellular concentration of Ca’+ and also that Ca2+ ionophore A23187 can promote ISC formation, and have used this data to support the hypothesis that Caz+ accumulation in the cells is an essential determinant of the changes leading to irreversible sickling (Eaton et al, 1973, 1978, 1979; Palek, 1973). This idea is not supported by our present data and that ofothers (Jensen et al, 1973; Clark et al, 1976) which shows that ISC formation can occur in the complete absence of Ca’+ in the incubation medium, even though Ca2+ may accelerate processes related to the formation of ISCs. These conflicting observations may be reconciled by supposing that Ca2+ ionophore A23187 or prolonged in vitro incubation (with or without Ca*+) cause a common biochemical change which is more directly related to ISC formation. One possibility is that the depletion of cellular ATP which occurs under both conditions is the central factor. Another suggestion, previously advanced by others, is that potassium chloride and water efflux, leading to cell shrinkage, is the important factor in both A23187 treated cells or those undergoing prolonged incubation (Glader & Sullivan, 1979; Clark eta!, 1980). Either of these changes may influence microvesicu- lation which could be more directly involved in the putative membrane lesion leading to irreversible sickling.

The relationship of ISCs produced in uitro experimentally to those identified in vivo as (generally) minor components of blood from sickle cell patients is not quite clear. Some authors have noted that in vivo ISCs are much less spiculed than are ISCs produced experimen- tally (Clark et al, 1976), and this may reflect the fact that the in vivo cells have lost their original spicules in the form of microvesicles. No such microvesicles were seen in any sample of fresh blood, possibly because these cell fragments are removed by phagocytic cells in the body.

In vivo ISCs have been found to be relatively depleted in fetal haemoglobin compared with the rest of the cell population and one possible explanation for this observation is that microvesicles released during the formation ofISCs are relatively enriched in HbF. Our results show that this does not apply to ISC formation in uitro: microvesicles showed no change in the ratio of HbF: HbS compared with the original cells.

ACKNOWLEDGMENTS

We wish to thank Dr Robert Hamill of Eli Lilly Co. for a gift of A23187 and Professor E. R. Huehns and Dr Ian Franklin of the Department of Clinical Haematology, University College London, for their interest in this work. We also appreciate the help of Professor J. M. White, Department of Haematology, Kings College Hospital Medical School, London, S.E.5. We gratefully acknowledge the financial support of the Medical Research Council.

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