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Morphological Changes of Human Erythrocytes Induced by Cholesterol Sulphate
Clinical Biochemistry, Vol. 31, No. 2, 73–79, 1998 Copyright © 1998 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/98 $19.00 1 .00
PII S0009-9120(97)00166-5
Morphological Changes of Human Erythrocytes Induced by Cholesterol Sulphate MARIA PRZYBYLSKA,1 MIL yOSZ FABER,1 ANDRZEJ ZABOROWSKI,1 JACEK S´WIE¸TOSL yAWSKI,2 1 and MARIA BRYSZEWSKA Institute of Biophysics, University of Lodz, Lodz, Poland, and 2Electron Microscopy Laboratory of Medical Academy in Lodz, Lodz, Poland
1
Objectives: Morphological alterations of human erythrocytes induced by cholesterol sulphate (5-cholesten-3b-ol sulphate, CS) were studied. Design and Methods: Influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy was studied. Results: In isotonic medium CS induces erythrocyte size and shape changes in dose- and time-dependent manner. Incubation (in vitro) of erythrocytes with CS concentrations from 4 3 1025 mol/dm3 to 8 3 1025 mol/dm3 led to a progressive sphero-echinocitic shape transformation accompanied by a cell size decrease. In contrast to this, for CS content equal to 1 3 1025 mol/dm3 the maintenance of the normal biconcave shape of red blood cells was observed. Conclusions: The results suggest that CS, similarly to numerous evaginating amphiphilic agents, induces a transformation of the erythrocyte normal discoid shape to echinocytic form. This effect may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of CS incorporated into the membrane. The echinocytic shape transformation of erythrocytes indicated that CS intercalates in the outer hemileaflet of the lipid bilayer leading to membrane externalization. Copyright © 1998 The Canadian Society of Clinical Chemists
KEY WORDS: cholesterol sulphate; erythrocyte; flow cytometry; scanning electron microscope.
Introduction terols are essential lipid constituents of all eukaryotic cells. These compounds not only play a major role in a variety of biochemical processes associated with biological membranes, but also serve as precursors for steroid hormones and bile acids. Literature has indicated that the most important sterol derivative is cholesterol sulphate (5-cholesten-3b-ol sulphate, CS)—a widespread compound found in mammalian body fluids and tissues (1– 4), especially highly elevated in sperm (5), endometrium (6,7), and keratinizing tissues such as epidermis (8,9), nails (10), hair (10,11), and hoof (12).
S
Correspondence: Dr. Maria Przybylska, Department of Biophysics, University of Lodz, 12/16 Banacha St, 90-237 Lodz, Poland. E-mail: [email protected] Manuscript received April 14, 1997; revised and accepted November 31, 1997. CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
Most of CS in animal cells resides in the plasma membrane, where it is believed to be an indispensable component, necessary to normal cellular function (13,14). In erythrocytes, CS participates in membrane stabilization and protects it against hypotonic (15) and thermally-induced (15,16) hemolysis. It has also been found to prevent Sendai virus from fusion to both human erythrocytes and liposomal membranes (17). In the upper layer of epidermis CS has been thought to play an important role in determining cohesion, desquamation, and permeability barrier function (18). CS may also be involved in the membrane modification of the spermatozoa acrosome during maturation process (5,19,20). It has been considered to be the crucial participant in the process of the embryo implantation into endometrium (6,7). It has also been suggested to be involved in the blood coagulation process by modulation of platelet function (21,22). In adrenal tissue CS serves as a precursor of 5-3b-hydroxysteroid sulphates (23, 24). Besides, CS may regulate lipid metabolic pathways related to growth and differentiation (25). Relatively large amount of CS in bile and feces suggests that it takes part in cholesterol excretion (26). CS has attracted much attention because of several serious diseases such as recessive X-linked ichthyosis (10,27,28), familial hypercholesterolemia (29), liver cirrhosis (29), homozygous sickle cell anemia (30), diabetes mellitus (31) and palmoplanar keratoderma (32) where abnormal CS levels have been reported. Although the molecular basis for the essential role of CS in animal cells has long been the object of intense interest, its function in the cell membranes is still not well understood. Therefore, the purpose of this study was to evaluate the influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy. 73
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ET AL.
Figure 1 — Scanning electron micrograph and scattering histograms of control erythrocytes incubated in PBS.
Materials and methods Chemicals of analytical grade were purchased from POCH (Gliwice, Poland). Cholesterol sulphate (5-cholesten-3b-ol sulphate) was from Steraloids (Wilton, NH, USA). BLOOD
PREPARATION
Blood samples from adult healthy subjects, obtained from the Blood Bank of Lodz, were taken into 3% trisodium citrate and centrifuged at 600 3 g for 10 minutes. Next red blood cells were washed three times with an equal volume of sodium phosphatebuffered saline (PBS: 0.150 mol/dm3 NaCl; 0.0019 mol/dm3, NaH2PO4; 0.0081 mol/dm3 Na2HPO4; pH 5 7.4) to remove plasma and white buffy coat. After washing, the packed erythrocytes were resuspended in medium at 1% hematocrit (1.5 3 108 cells/cm3). To tubes containing 3 cm3 of this cell suspension, CS in ethanol was added to give final concentrations from 0.4 3 1025 mol/dm3 to 8 3 1025 mol/dm3. To the other set of tubes the same volume of ethanol was added. The final concentration of ethanol, equal in all tubes, was 1%. Additionally, control samples without ethanol were prepared. Next, blood samples were incubated at room temperature for 0.5, 1.0, 1.5, or 2.0 hours, respectively. After this time each of these samples was centrifuged at 600 3 g for 10 minutes and resuspended in the same medium. All measurements of this study were performed on freshly prepared erythrocytes. FLOW
CYTOMETRY
Red blood cells suspensions (0.5 cm3) containing approximately 106 cells/cm3 were analyzed using a Scatron Argus (Norway) arc lamp-based compact flow cytometer, with simultaneous separate detection at low angle (LS1) and right angle (LS2). The light scattered near the forward direction (low angle) is expected to be proportional to the size (volume) of the particle and is independent of cell refractive index and shape, whereas scattering at the right angle depends on cell shape and internal properties of the scattering particles (33,34). LS1/ LS2 is a dual parameter contour plot histogram proportional to the total cell diversity. For each set 74
of histograms the percentage of altered cells and peak position (P) was obtained from values collected by using a standard computer programme for the Argus cytometer. RED
BLOOD CELL SHAPE OBSERVATION
For scanning electron microscope observations, erythrocytes were washed twice with an equal volume of sodium-potassium phosphate buffer (0.164 mol/dm3 Na2HPO4, 0.036 mol/dm3 KH2PO4; pH 5 7.4) and were fixed with 1% glutaraldehyde in the same buffer to achieve final haematocrit of about 50%. Fixed cells were allowed to settle on standard microscopic cover glasses. After 1.0 h cover glasses were washed twice with phosphate buffer. Subsequently samples were dehydrated with successive washes in ascending ethanol series (30%, 50%, 70%, 85%, 95%; v/v), in 100% acetone and finally dried with CO2 in the triple-point. Finally, erythrocytes were coated with gold-palladium and examined in a Stereoscan 600 scanning electron microscope (Cambridge Instruments, UK). In each sample 100 erythrocytes were classified and mean morphological index (MI) was calculated. Echinocytes were assigned a morphology score of 1–5 and discocytes 0, based on Bessi’s nomenclature for classification of red blood cells shapes (35). STATISTICAL
ANALYSIS
Data are expressed as means 6 SD. For further analysis of erythrocyte morphology, hierarchical analysis of variance was used. Before this procedure data were transformed as arc sin =xi (LS1, LS2, and LS1/LS2) or log xi (peak position) function (36). Results FLOW
CYTOMETRY
Series of scans obtained by flow cytometry measurements reflect the development of changes in erythrocyte size, shape and membrane surface morphology. Typical scattering histograms for control erythrocytes are presented in Figure 1. The curve plotted on LS1 histogram and on LS2 histogram are smooth, sharp, and almost symmetric. The surface contour plotted on LS1/LS2 histogram is greatly concentrated. CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
CHOLESTEROL SULPHATE-INDUCED CHANGES IN HUMAN RED BLOOD CELLS
Figure 2 — Scattering histograms of erythrocytes incubated in PBS containing cholesterol sulphate, (A) 0.4 3 1025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3.
For CS content equal to 1 3 1025 mol/dm3, curves plotted on both scattering histograms, the dual parameter contour plot and peak position (P) in comparison with control ones remain almost unchanged even for the longest time (2h) of incubation (Figures 1 and 2). For lower (0.4 3 1025 mol/dm3) and higher (4 – 8 3 1025 mol/dm3) (Figure 2) CS concentrations the curves become more flat, jagged, asymmetric, and eventually double-peaked, and the peak position (P) is significantly changed. The extension of changes is CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
especially significant for the highest (8 3 1025 mol/dm3) CS concentration used (Figure 2). The diversity in the scattering erythrocyte properties, expressed as a percentage of altered cells and peak position (P) induced by CS as a function of time and CS concentration was summarized on Figure 3. Hierarchical analysis of variance (with time of incubation as a nested factor within cholesterol sulphate concentration) showed that both factor have significant effects on the morphology of human erythrocytes (Table 1). 75
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Figure 3 — Flow cytometric analysis of cholesterol sulphate effect on erythrocytes morphological parameters (A) 0.4 3 1025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3 of cholesterol sulphate). Data are expressed as mean 6 SD (n 5 4).
RED
forms with intercellular bridges indicating a sticky nature of the cell surfaces and leads progressively to a decrease of a cell size (Figure 4). The greatest degree of erythrocyte shape and size modification has occurred at CS concentration of 8 3 1025 mol/dm3, especially for longer incubation time (2 h). Only for CS concentration equal to 1 3 1025 mol/dm3 the maintenance of the normal
BLOOD CELL SHAPE OBSERVATION
As it is shown in Figure 4, erythrocytes subjected to CS concentration of 0.4 3 1025 mol/dm3 for 0.5 to 2.0 h underwent only few morphological changes, in comparison with control cells (Figure 1). The increasing CS content transforms red blood cells from discocytes into sphero-echinocitic
TABLE 1 Hierarchical Analysis of Variance of Erythrocyte Morphological Features LS1 Source of Variation CS concentration Time of incubation Residual
MS 6068.8 250.0 14.3
df 3 12 48
LS2 F
MS a
424.0 17.5a
5758.0 231.9 19.8
df 3 12 48
LS1/LS2 F
MS a
290.5 11.7a
5059.0 204.3 16.3
df 3 12 48
Peak Position F a
310.8 12.6a
MS
df
F
0.445 0.028 0.001
3 12 48
405.3a 25.5a
Data were transformed as arcsin =xi (LS1, LS2, and LS1/LS2) or log xi (peak position (P). p , 0.001.
a
76
CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
CHOLESTEROL SULPHATE-INDUCED CHANGES IN HUMAN RED BLOOD CELLS
Figure 4 — Scanning electron micrographs of erythrocytes incubated in PBS containing cholesterol sulphate, (A) 0.4 3 1025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3.
smooth biconcave shape of red blood cells was observed (Figure 4). The degree of erythrocyte echinocytic transformations, expressed as morphological indexes (MI), for increasing incubation times and CS concentrations are summarised in Figure 5.
Flow cytometrical scans reflect these changes showing significant differences in erythrocyte size (volume), shape and surface structure upon the
Discussion The normal human erythrocyte is a flexible biconcave disk. Numerous studies have shown that erythrocytes respond to various treatments by altering their morphological features (37– 40). The comparison of series of scattering histograms obtained by flow cytometry (Figures 1 and 2) and corresponding SEM images (Figures 1– 4) indicates that in isotonic solution cholesterol sulphate, similarly to a variety of anionic amphiphilic agents (37– 40), causes erythrocyte size and shape changes, in dose- and time-dependent manner (Figure 3). We found that the effect of this compound on erythrocyte stability is distinctly biphasic. Only in a narrow range of CS content (1 3 1025 mol/dm3), equal to its physiological levels (14), erythrocytes maintained their native, smooth, biconcave shape, even for longer incubation time (2 h). Contrary to this, the incubation of red blood cells with lower (0.4 3 1025 mol/dm3) and higher (4 – 8 3 1025 mol/dm3) CS concentrations, promotes substantial morphological alterations. CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
Figure 5 — The degree of erythrocyte echinocytic transformations, expressed as the morphological indexes (MI), obtained with increasing CS concentration. Data are expressed as mean 6 SD. 77
PRZYBYLSKA
influence of CS. Jagged, asymmetric and doublepeaked scattering curves suggesting the existence of two subpopulations of cells (Figure 2) are well confirmed by the results obtained by SEM showing the formation of spicules, vesicles and numerous intercellular links (Figure 4). It is noteworthy that these morphological changes take place at CS concentration above physiological level, but in the range found in patients suffering from certain diseases such as recessive X-linked ichthyosis, where CS concentration in plasma varies from 5.8 3 1025 to 8.6 3 1025 mol/dm3 (41). Morphological alterations induced by CS can be explained, in terms of Sheetz and Singer (42) bilayer couple hypothesis. According to it, shape alterations arise from a differential expansion of amphipatic molecules into two monolayers of cell membranes. Echinocytogenic amphiphiles such as CS, are thought to be preferentially intercalated in the outer hemileaflet of the lipid bilayer and, at sublytic concentrations, cause red blood cells transformation from discoid to echinocytic forms (42). However, for low concentrations (0.4 –1 3 1025 mol/dm3) this sterol does not involve substantial perturbation of the plasma membrane, which may be, at least partly, explained by the ability of this amphipathic molecule to increase membrane surface pressure and hydration (due to the presence of ionised hydroxyl group in physiological conditions) (43). Our results are in agreement with earlier studies on the interaction of amphiphiles with the erythrocyte membrane, which have suggested that the erythrocyte membrane can incorporate definite amount of foreign amphiphiles without losing its integrity and barrier properties (39). The interesting point of our observations is that the comparison of the degree of echinocytic transformation, expressed in terms of the morphological index (Figure 5) with data which present the percentage of altered cells and peak position (P) positions (Figure 3) obtained by cytometric measurements, leads to the conclusion that both methods provide convergent information. It is worth emphasizing that the cytometric method seems to be far better than the others. It almost completely allows to avoid drastic, time consuming sample preparation, which may alter red blood cells morphological features and provides significantly better data recurrence (compare standard deviation values on Figures 3 and 5). In conclusion, it may be considered as a useful method for carrying out measurements of various agents effect on cell morphology and membrane properties. In this context, it is important to note that indicating a biphasic behavior cholesterol sulphate may have particular relevance to many phenomena in cell biology that involve shape changes, including cell locomotion, cell-cell and cell-virus fusion, secretion, phagocytosis and the others. Due to its unique properties CS may be considered to be a potent modulator of liposome stability essential for drug 78
ET AL.
dosage control and release properties in liposomebased therapeutic agent (44). Further study of molecular mechanisms involved in a protective ability of CS against cell membrane destabilization may help develop a new target for antiviral therapy (17). Acknowledgement The authors wishes to thank Dr. Miroslyaw Przybylski for his advice and help in statistical analysis of data.
References 1. Moser HW, Moser AB, Orr J. Preliminary observations on the occurrence of cholesterol sulfate in man. Biochim Biophys Acta 1966; 116: 146 –55. 2. Bleau G, Chapdelaine A, Roberts KD. The assay of cholesterol sulfate in biological material by enzymatic radioisotopic displacement. Can J Biochem 1972; 50: 277– 86. 3. Iwamori M, Moser HW, Kishimoto Y. Cholesterol sulfate in rat tissues. Tissue distribution, developmental change and brain subcellular localization. Biochim Biophys Acta 1976; 441: 268 –79. 4. Veares MP, Evershed RP, Prescott MC, Goad LJ. Quantitative determination of cholesterol sulphate in plasma by stable isotope dilution fast atom bombardment mass spectrometry. Biomed Environ Mass Spectrom 1990; 19: 583– 8. 5. Langlais J, Zollinger M, Plante L, Chapdelaine A, Bleau G, Roberts KD. Localization of cholesteryl sulfate in human spermatozoa support of a hypothesis for the mechanism of capacitation. Proc Natl Acad Sci USA 1981; 78: 7266 –70. 6. Momoeda M, Taketani Y, Mizuno M, Iwamori M, Nagai Y. Characteristic expression of cholesterol sulphate in rabbit endometrium during the implantation period. Biochem Biophys Res Commun 1991; 178: 145–50. 7. Nicollier M, Beck L, Mahfoudi A, Coosemans V, Adessi GL. Effect of progesterone on hydrophobic cell-associated proteoglycans bound to cholesterol sulfate in glandular epithelial cells of guinea-pig endometrium. Biochim Biophys Acta 1994; 13: 125–31. 8. Serizawa S, Osawa K, Togashi K, et al. Relationship between cholesterol sulfate and intercellular cohesion of the stratum corneum: Demonstration using a pushpull meter and an improved high-performance tinlayer chromatographic separation system of all major stratum corneum lipids. J Invest Dermatol 1992; 99: 232– 6. 9. Wertz PW. Epidermal lipids. Semin Dermatol 1992; 11: 106 –13. 10. Serizawa S, Nagai T, Ito M, Sato Y. Cholesterol sulphate levels in the hair and nails of patients with recessive X-linked ichthosis. Clin Exp Dermatol 1990; 15: 13–15. 11. Brosche T, Gollwitzer J, Platt D. Cholesterol and cholesterol sulphate concentration in the cell membrane complex of human scalp hair—a biomarker of aging? Arch Gerontol Geriatr Suppl 1994; 4: 19 –30. 12. Wertz PW, Downing DT. Cholesterol sulfate: the major polar lipid of horse hoof. J Lipid Res 1984; 25: 1320 –3. 13. Bleau G, Lalumierre´ G, Chapdelaine A, Roberts KD. Red cell surface structure. Stabilization by cholesterol CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
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sulfate as evidenced by scanning electron microscopy. Biochim Biophys Acta 1975; 375: 220 –3. Bleau G, Bodley FH, Longprre´ J, Chapdelaine A, Roberts KD. Cholesterol sulfate. I. Occurrence and possible biological function as an amphipathic lipid in the membrane of the human erythrocyte. Biochim Biophys Acta 1974; 352: 1–9. Bryszewska M, Koter M, Epand RM. Effect of cholesterol sulphate on haemolysis of human erythrocytes. Med Sci Res 1991; 19: 791–2. Przybylska M, Bryszewska M, Epand RM. Effect of cholesterol sulphate on the thermosensitivity and fluidity of human erythrocytes. Biomed Lett 1994; 49: 113–17. Cheetham JJ, Epand RM, Andrews M, Flanagan TD. Cholesterol sulfate inhibits the fusion of Sendai virus to biological and model membranes. J Biol Chem 1990; 265: 12404 –9. Williams ML, Rutherford SL, Feingold KR. Effects of cholesterol sulfate on lipid metabolism in cultured human keratinocytes and fibroblasts. J Lipid Res 1987; 28: 955– 67. Cheetham JJ, Chen RJB, Epand RM. Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction. Biochim Biophys Acta 1990; 1024: 367–72. Davis BK, Byrne R, Bedigian K. Studies on the mechanism of capacitation. Albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells. Proc Natl Acad Sci USA 1980; 77: 1546–1550. Blache D, Becchi M, Davignon J. Occurrence and biological effects of cholesterol sulfate on blood-platelets. Biochim Biophys Acta 1995; 1259: 291– 6. Blache D. Enhanced arachidonic acid and calcium metabolism in cholesteryl sulfate-enrched rat platelets. J Lipid Med Cell Signal 1996; 13: 127–38. Roberts KD, Bandi L, Calvin HI, Drucker WD, Lieberman S. Evidence that steroid sulfates serve as biosynthetic intermediates. IV. Conversion of cholesterol sulfate in vivo to urinary C19 and C21 steroid sulfates. Biochem 1964; 15: 1983–1988. Roberts KD, Bandi L, Calvin HI, Drucker WD, Lieberman S. Evidence that steroid sulfate is a precursor of steroid hormones. J Am Chem Soc 1964; 86: 958 –9. Woscholski R, Kodaki T, Palmer RH, Waterfield MD, Parker PJ. Modulation of the substrate specificity of the mammalian phosphatidiloinositol 3-kinase by cholesterol sulfate and sulfatide. Biochemistry 1995; 36: 11489 –93. Chen L, Imperato TJ, Bolt RJ. Enzymatic sulfation of bile salt by sulfotransferase from rat kidney. Biochim Biophys Acta 1978; 522: 443–51. Muskiet FAJ, Jansen G, Wolthers BG, MarinkovicIlsen A, Van Voorst Vader P. Gas-chromatographic determination of cholesterol sulfate in plasma and erythrocytes, for the diagnosis of recessive X-linked ichthyosis. Clin Chem 1983; 29: 1404 –7. Shwayder T, Ott F. All about ichthyosis. Ped Clin North Am 1991; 38: 835–57.
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29. Tamasawa N, Tamasawa A, Takebe K. Higher levels of plasma cholesterol sulphate in patients with liver cirrhosis and hipercholesterolemia. Lipids 1993; 28: 833– 6. 30. Muskiet FD, Muskiet FAJ. Lipids, fatty acids and trace elements in plasma and erythrocytes of pediatric patients with homozygous sickle cell disease. Clin Chim Acta 1984; 142: 1–10. 31. Przybylska M, Bryszewska M, Nowicka U, Szosland K, Ke¸dziora J, Epand RM. Estimation of cholesterol sulphate in blood plasma and in erythrocytes membranes from individuals with Down’s syndrome or diabetes mellitus type I. Clin Biochem 1995; 28(6): 593–7. 32. Barlag KE, Goerz G, Ruzicka T, Schurer NY. Palmoplanar keratoderma with an unusual composition of stratum-corneum and serum sterol derivatives—a new entity. Br J Dermatol 1995; 133: 639 – 43. 33. Salzman GC, Singham SB, Johnston RG, Bohren CF. Light scattering and cytometry. In: Flow Cytometry and Sorting. (Eds. Melamed MR, Mullaney PF, Mendelsohn MR), pp. 81–107. New York: Wiley-Liss, Inc. 1990. 34. Visser JWM, van den Engh GJ, van Bekkum DW. Light scattering properties of murine hemopoetic cells. Blood Cells 1980; 6: 391– 407. 35. Bessis M. Red cell shapes: an illustrated classification and its rationale. In: Red Cell Shape. (Eds. Bessis M, Weed RI, Le Blond PF), pp. 1–23. New York: SpringerVerlag, 1973. 36. Zar JH. Biostatistical analysis. London: PrenticeHall, 1984. 37. Seeman P, Kwant WO, Sauks T, Argent W. Membrane expansion of intact erythrocytes by anesthetics. Biochim Biophys Acta 1969; 183: 490 – 8. 38. Seeman P. The membrane actions of anesthetics and tranquilizers. Pharmacol Rev 1972; 24: 583– 633. 39. Ha¨gerstrand H, Isoma B. Vesiculation induced by amphiphiles in erythrocytes. Biochim Biophys Acta 1989; 982: 179 – 86. 40. Reinhart WH, Rohner F. Effect of amiodarone on erythrocyte shape and membrane properties. Clin Sci 1990; 79: 387–91. 41. Bergner EA, Shapiro LJ. Increased cholesterol sulfate in plasma and red blood cell membranes of steroid sulfatase deficient patients. J Clin Endocrinol Metab 1981; 53: 221–3. 42. Sheetz MP, Singer SJ. Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc Natl Acad Sci USA 1974; 71: 4457– 61. 43. Faure C, Tranchant JF, Dufourc EJ. Comparative effects of cholesterol and cholesterol sulfate on hydration and ordering of dimyristoylphosphatidylo-choline membranes. Biophys J 1996; 70: 1380 –90. 44. Viani P, Cervato G, Gatti P, Cestaro B. Plasma dependent pH sensitivity of liposomes containing sulfatide. Biochim Biophys Acta 1993; 8: 73– 80.
79
PII S0009-9120(97)00166-5
Morphological Changes of Human Erythrocytes Induced by Cholesterol Sulphate MARIA PRZYBYLSKA,1 MIL yOSZ FABER,1 ANDRZEJ ZABOROWSKI,1 JACEK S´WIE¸TOSL yAWSKI,2 1 and MARIA BRYSZEWSKA Institute of Biophysics, University of Lodz, Lodz, Poland, and 2Electron Microscopy Laboratory of Medical Academy in Lodz, Lodz, Poland
1
Objectives: Morphological alterations of human erythrocytes induced by cholesterol sulphate (5-cholesten-3b-ol sulphate, CS) were studied. Design and Methods: Influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy was studied. Results: In isotonic medium CS induces erythrocyte size and shape changes in dose- and time-dependent manner. Incubation (in vitro) of erythrocytes with CS concentrations from 4 3 1025 mol/dm3 to 8 3 1025 mol/dm3 led to a progressive sphero-echinocitic shape transformation accompanied by a cell size decrease. In contrast to this, for CS content equal to 1 3 1025 mol/dm3 the maintenance of the normal biconcave shape of red blood cells was observed. Conclusions: The results suggest that CS, similarly to numerous evaginating amphiphilic agents, induces a transformation of the erythrocyte normal discoid shape to echinocytic form. This effect may be caused, at least partly, by an asymmetric expansion of the membrane lipid bilayer due to asymmetric distribution of CS incorporated into the membrane. The echinocytic shape transformation of erythrocytes indicated that CS intercalates in the outer hemileaflet of the lipid bilayer leading to membrane externalization. Copyright © 1998 The Canadian Society of Clinical Chemists
KEY WORDS: cholesterol sulphate; erythrocyte; flow cytometry; scanning electron microscope.
Introduction terols are essential lipid constituents of all eukaryotic cells. These compounds not only play a major role in a variety of biochemical processes associated with biological membranes, but also serve as precursors for steroid hormones and bile acids. Literature has indicated that the most important sterol derivative is cholesterol sulphate (5-cholesten-3b-ol sulphate, CS)—a widespread compound found in mammalian body fluids and tissues (1– 4), especially highly elevated in sperm (5), endometrium (6,7), and keratinizing tissues such as epidermis (8,9), nails (10), hair (10,11), and hoof (12).
S
Correspondence: Dr. Maria Przybylska, Department of Biophysics, University of Lodz, 12/16 Banacha St, 90-237 Lodz, Poland. E-mail: [email protected] Manuscript received April 14, 1997; revised and accepted November 31, 1997. CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
Most of CS in animal cells resides in the plasma membrane, where it is believed to be an indispensable component, necessary to normal cellular function (13,14). In erythrocytes, CS participates in membrane stabilization and protects it against hypotonic (15) and thermally-induced (15,16) hemolysis. It has also been found to prevent Sendai virus from fusion to both human erythrocytes and liposomal membranes (17). In the upper layer of epidermis CS has been thought to play an important role in determining cohesion, desquamation, and permeability barrier function (18). CS may also be involved in the membrane modification of the spermatozoa acrosome during maturation process (5,19,20). It has been considered to be the crucial participant in the process of the embryo implantation into endometrium (6,7). It has also been suggested to be involved in the blood coagulation process by modulation of platelet function (21,22). In adrenal tissue CS serves as a precursor of 5-3b-hydroxysteroid sulphates (23, 24). Besides, CS may regulate lipid metabolic pathways related to growth and differentiation (25). Relatively large amount of CS in bile and feces suggests that it takes part in cholesterol excretion (26). CS has attracted much attention because of several serious diseases such as recessive X-linked ichthyosis (10,27,28), familial hypercholesterolemia (29), liver cirrhosis (29), homozygous sickle cell anemia (30), diabetes mellitus (31) and palmoplanar keratoderma (32) where abnormal CS levels have been reported. Although the molecular basis for the essential role of CS in animal cells has long been the object of intense interest, its function in the cell membranes is still not well understood. Therefore, the purpose of this study was to evaluate the influence of CS on red blood cell stability (in isotonic conditions) by simultaneous application of flow cytometry and scanning electron microscopy. 73
PRZYBYLSKA
ET AL.
Figure 1 — Scanning electron micrograph and scattering histograms of control erythrocytes incubated in PBS.
Materials and methods Chemicals of analytical grade were purchased from POCH (Gliwice, Poland). Cholesterol sulphate (5-cholesten-3b-ol sulphate) was from Steraloids (Wilton, NH, USA). BLOOD
PREPARATION
Blood samples from adult healthy subjects, obtained from the Blood Bank of Lodz, were taken into 3% trisodium citrate and centrifuged at 600 3 g for 10 minutes. Next red blood cells were washed three times with an equal volume of sodium phosphatebuffered saline (PBS: 0.150 mol/dm3 NaCl; 0.0019 mol/dm3, NaH2PO4; 0.0081 mol/dm3 Na2HPO4; pH 5 7.4) to remove plasma and white buffy coat. After washing, the packed erythrocytes were resuspended in medium at 1% hematocrit (1.5 3 108 cells/cm3). To tubes containing 3 cm3 of this cell suspension, CS in ethanol was added to give final concentrations from 0.4 3 1025 mol/dm3 to 8 3 1025 mol/dm3. To the other set of tubes the same volume of ethanol was added. The final concentration of ethanol, equal in all tubes, was 1%. Additionally, control samples without ethanol were prepared. Next, blood samples were incubated at room temperature for 0.5, 1.0, 1.5, or 2.0 hours, respectively. After this time each of these samples was centrifuged at 600 3 g for 10 minutes and resuspended in the same medium. All measurements of this study were performed on freshly prepared erythrocytes. FLOW
CYTOMETRY
Red blood cells suspensions (0.5 cm3) containing approximately 106 cells/cm3 were analyzed using a Scatron Argus (Norway) arc lamp-based compact flow cytometer, with simultaneous separate detection at low angle (LS1) and right angle (LS2). The light scattered near the forward direction (low angle) is expected to be proportional to the size (volume) of the particle and is independent of cell refractive index and shape, whereas scattering at the right angle depends on cell shape and internal properties of the scattering particles (33,34). LS1/ LS2 is a dual parameter contour plot histogram proportional to the total cell diversity. For each set 74
of histograms the percentage of altered cells and peak position (P) was obtained from values collected by using a standard computer programme for the Argus cytometer. RED
BLOOD CELL SHAPE OBSERVATION
For scanning electron microscope observations, erythrocytes were washed twice with an equal volume of sodium-potassium phosphate buffer (0.164 mol/dm3 Na2HPO4, 0.036 mol/dm3 KH2PO4; pH 5 7.4) and were fixed with 1% glutaraldehyde in the same buffer to achieve final haematocrit of about 50%. Fixed cells were allowed to settle on standard microscopic cover glasses. After 1.0 h cover glasses were washed twice with phosphate buffer. Subsequently samples were dehydrated with successive washes in ascending ethanol series (30%, 50%, 70%, 85%, 95%; v/v), in 100% acetone and finally dried with CO2 in the triple-point. Finally, erythrocytes were coated with gold-palladium and examined in a Stereoscan 600 scanning electron microscope (Cambridge Instruments, UK). In each sample 100 erythrocytes were classified and mean morphological index (MI) was calculated. Echinocytes were assigned a morphology score of 1–5 and discocytes 0, based on Bessi’s nomenclature for classification of red blood cells shapes (35). STATISTICAL
ANALYSIS
Data are expressed as means 6 SD. For further analysis of erythrocyte morphology, hierarchical analysis of variance was used. Before this procedure data were transformed as arc sin =xi (LS1, LS2, and LS1/LS2) or log xi (peak position) function (36). Results FLOW
CYTOMETRY
Series of scans obtained by flow cytometry measurements reflect the development of changes in erythrocyte size, shape and membrane surface morphology. Typical scattering histograms for control erythrocytes are presented in Figure 1. The curve plotted on LS1 histogram and on LS2 histogram are smooth, sharp, and almost symmetric. The surface contour plotted on LS1/LS2 histogram is greatly concentrated. CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
CHOLESTEROL SULPHATE-INDUCED CHANGES IN HUMAN RED BLOOD CELLS
Figure 2 — Scattering histograms of erythrocytes incubated in PBS containing cholesterol sulphate, (A) 0.4 3 1025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3.
For CS content equal to 1 3 1025 mol/dm3, curves plotted on both scattering histograms, the dual parameter contour plot and peak position (P) in comparison with control ones remain almost unchanged even for the longest time (2h) of incubation (Figures 1 and 2). For lower (0.4 3 1025 mol/dm3) and higher (4 – 8 3 1025 mol/dm3) (Figure 2) CS concentrations the curves become more flat, jagged, asymmetric, and eventually double-peaked, and the peak position (P) is significantly changed. The extension of changes is CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
especially significant for the highest (8 3 1025 mol/dm3) CS concentration used (Figure 2). The diversity in the scattering erythrocyte properties, expressed as a percentage of altered cells and peak position (P) induced by CS as a function of time and CS concentration was summarized on Figure 3. Hierarchical analysis of variance (with time of incubation as a nested factor within cholesterol sulphate concentration) showed that both factor have significant effects on the morphology of human erythrocytes (Table 1). 75
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ET AL.
Figure 3 — Flow cytometric analysis of cholesterol sulphate effect on erythrocytes morphological parameters (A) 0.4 3 1025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3 of cholesterol sulphate). Data are expressed as mean 6 SD (n 5 4).
RED
forms with intercellular bridges indicating a sticky nature of the cell surfaces and leads progressively to a decrease of a cell size (Figure 4). The greatest degree of erythrocyte shape and size modification has occurred at CS concentration of 8 3 1025 mol/dm3, especially for longer incubation time (2 h). Only for CS concentration equal to 1 3 1025 mol/dm3 the maintenance of the normal
BLOOD CELL SHAPE OBSERVATION
As it is shown in Figure 4, erythrocytes subjected to CS concentration of 0.4 3 1025 mol/dm3 for 0.5 to 2.0 h underwent only few morphological changes, in comparison with control cells (Figure 1). The increasing CS content transforms red blood cells from discocytes into sphero-echinocitic
TABLE 1 Hierarchical Analysis of Variance of Erythrocyte Morphological Features LS1 Source of Variation CS concentration Time of incubation Residual
MS 6068.8 250.0 14.3
df 3 12 48
LS2 F
MS a
424.0 17.5a
5758.0 231.9 19.8
df 3 12 48
LS1/LS2 F
MS a
290.5 11.7a
5059.0 204.3 16.3
df 3 12 48
Peak Position F a
310.8 12.6a
MS
df
F
0.445 0.028 0.001
3 12 48
405.3a 25.5a
Data were transformed as arcsin =xi (LS1, LS2, and LS1/LS2) or log xi (peak position (P). p , 0.001.
a
76
CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
CHOLESTEROL SULPHATE-INDUCED CHANGES IN HUMAN RED BLOOD CELLS
Figure 4 — Scanning electron micrographs of erythrocytes incubated in PBS containing cholesterol sulphate, (A) 0.4 3 1025 mol/dm3, (B) 1 3 1025 mol/dm3, (C) 4 3 1025 mol/dm3, (D) 8 3 1025 mol/dm3.
smooth biconcave shape of red blood cells was observed (Figure 4). The degree of erythrocyte echinocytic transformations, expressed as morphological indexes (MI), for increasing incubation times and CS concentrations are summarised in Figure 5.
Flow cytometrical scans reflect these changes showing significant differences in erythrocyte size (volume), shape and surface structure upon the
Discussion The normal human erythrocyte is a flexible biconcave disk. Numerous studies have shown that erythrocytes respond to various treatments by altering their morphological features (37– 40). The comparison of series of scattering histograms obtained by flow cytometry (Figures 1 and 2) and corresponding SEM images (Figures 1– 4) indicates that in isotonic solution cholesterol sulphate, similarly to a variety of anionic amphiphilic agents (37– 40), causes erythrocyte size and shape changes, in dose- and time-dependent manner (Figure 3). We found that the effect of this compound on erythrocyte stability is distinctly biphasic. Only in a narrow range of CS content (1 3 1025 mol/dm3), equal to its physiological levels (14), erythrocytes maintained their native, smooth, biconcave shape, even for longer incubation time (2 h). Contrary to this, the incubation of red blood cells with lower (0.4 3 1025 mol/dm3) and higher (4 – 8 3 1025 mol/dm3) CS concentrations, promotes substantial morphological alterations. CLINICAL BIOCHEMISTRY, VOLUME 31, MARCH 1998
Figure 5 — The degree of erythrocyte echinocytic transformations, expressed as the morphological indexes (MI), obtained with increasing CS concentration. Data are expressed as mean 6 SD. 77
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influence of CS. Jagged, asymmetric and doublepeaked scattering curves suggesting the existence of two subpopulations of cells (Figure 2) are well confirmed by the results obtained by SEM showing the formation of spicules, vesicles and numerous intercellular links (Figure 4). It is noteworthy that these morphological changes take place at CS concentration above physiological level, but in the range found in patients suffering from certain diseases such as recessive X-linked ichthyosis, where CS concentration in plasma varies from 5.8 3 1025 to 8.6 3 1025 mol/dm3 (41). Morphological alterations induced by CS can be explained, in terms of Sheetz and Singer (42) bilayer couple hypothesis. According to it, shape alterations arise from a differential expansion of amphipatic molecules into two monolayers of cell membranes. Echinocytogenic amphiphiles such as CS, are thought to be preferentially intercalated in the outer hemileaflet of the lipid bilayer and, at sublytic concentrations, cause red blood cells transformation from discoid to echinocytic forms (42). However, for low concentrations (0.4 –1 3 1025 mol/dm3) this sterol does not involve substantial perturbation of the plasma membrane, which may be, at least partly, explained by the ability of this amphipathic molecule to increase membrane surface pressure and hydration (due to the presence of ionised hydroxyl group in physiological conditions) (43). Our results are in agreement with earlier studies on the interaction of amphiphiles with the erythrocyte membrane, which have suggested that the erythrocyte membrane can incorporate definite amount of foreign amphiphiles without losing its integrity and barrier properties (39). The interesting point of our observations is that the comparison of the degree of echinocytic transformation, expressed in terms of the morphological index (Figure 5) with data which present the percentage of altered cells and peak position (P) positions (Figure 3) obtained by cytometric measurements, leads to the conclusion that both methods provide convergent information. It is worth emphasizing that the cytometric method seems to be far better than the others. It almost completely allows to avoid drastic, time consuming sample preparation, which may alter red blood cells morphological features and provides significantly better data recurrence (compare standard deviation values on Figures 3 and 5). In conclusion, it may be considered as a useful method for carrying out measurements of various agents effect on cell morphology and membrane properties. In this context, it is important to note that indicating a biphasic behavior cholesterol sulphate may have particular relevance to many phenomena in cell biology that involve shape changes, including cell locomotion, cell-cell and cell-virus fusion, secretion, phagocytosis and the others. Due to its unique properties CS may be considered to be a potent modulator of liposome stability essential for drug 78
ET AL.
dosage control and release properties in liposomebased therapeutic agent (44). Further study of molecular mechanisms involved in a protective ability of CS against cell membrane destabilization may help develop a new target for antiviral therapy (17). Acknowledgement The authors wishes to thank Dr. Miroslyaw Przybylski for his advice and help in statistical analysis of data.
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