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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4373-4378
The Efficacy of Reducing Agents or Antioxidants in Blocking the
Formation of Dense Cells and Irreversibly Sickled Cells In Vitro
By
Xunda A. Gibson,
Archil Shartava,
Jonah McIntyre,
Carlos A. Monteiro,
Yalin Zhang,
Arvind Shah,
Naomi F. Campbell, and
Steven
R. Goodman
From the Departments of Structural and Cellular Biology, Mathematics
and Statistics, Chemistry, and USA Comprehensive Sickle Cell Center,
University of South Alabama College of Medicine, Mobile, AL.
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ABSTRACT |
We show that N-acetylcysteine (NAC) has the ability to cause
statistically significant diminishment in the in vitro formation of
irreversibly sickled cells (ISCs) at concentrations greater than 250 µmol/L. Other antioxidants, approved for human use (cysteamine, succimer, dimercaprol), were not efficacious. NAC had the ability to
cause statistically significant conversion of ISCs formed in vivo back
to the biconcave shape. NAC was also shown to reduce the formation of
dense cells and increase the available thiols in  -actin. We showed
that diminishing reduced glutathione (GSH), by treatment with
1-chloro-2,4-dinitrobenzene, resulted in increased dense cells. We
conclude the NAC blocks dense cell formation and ISC formation by
targeting channels involved in cellular dehydration and -actin,
respectively. The efficacy of NAC is probably due to its combined
antioxidant activity and ability to increase intracellular GSH.
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INTRODUCTION |
BLOOD FROM HOMOZYGOUS sickle cell (SS)
patients can be separated by density gradient sedimentation into
morphologically and physiologically distinct red blood cell (RBC)
classes.1 The highest density class of RBCs includes
irreversibly sickled cells (ISCs) (60% to 85%) that retain a sickled
shape in well-oxygenated blood where the sickle cell hemoglobin (HbS)
is depolymerized. These ISCs represent up to 45% of the circulating
RBCs in SS blood and they are poorly deformable, of short life span,
and have lower levels of fetal hemoglobin than do reversibly sickled
cells (RSCs).2 During the course of vasoocclusion, the
highest density class of SS RBCs is selectively trapped in the
microvasculature.3,4 The dense cell population appears to
block the narrowed lumen of vessels lined primarily with the more
adherent lower density RBCs.5 Hemolysis is correlated with
the dense cell population, which contains RBCs that have the greatest
propensity to form polymer, are most susceptible to shear stress, and
appear to play an essential role in vasoocclusion.6-8
The spectrin membrane skeleton controls the shape, elasticity, and
flexibility of the RBC.9 The major essential components of
the membrane skeleton are spectrin, protein 4.1, and
actin.10 We have recently shown that a posttranslational
modification in ISC -actin, in which a disulfide bridge forms
between cysteine 284 and cysteine 373,11,12 leads to an ISC
membrane skeleton, which can only slowly disassemble at
37°C.11,13 The slow disassembly is caused by altered
ISC actin polymerization-depolymerization kinetics.14 Based
on this evidence, we proposed that the inability of ISC membrane
skeletal proteins to reassemble at 37°C leads to the ISC
skeleton's inability to remodel.15 Furthermore, we proposed that membrane permeable reducing agents or antioxidants, which
can block the -actin disulfide bridge from forming, should be able
to inhibit ISC formation.15
Jensen et al16 found that ISCs could be generated in vitro
when SS RBCs were kept under deoxygenation conditions for 24 hours.
Based on this finding, Ohnishi et al17 showed that ISCs can
be efficiently generated in vitro by deoxygenation-reoxygenation cycling of RSCs. We have made use of the methodology to test the efficacy of various clinically relevant reducing agents or antioxidants to block ISC formation in vitro. In this study, we show that (1) N-acetylcysteine has the ability to block ISC formation at
pharmacologically achievable dosages; (2) N-acetylcysteine can convert
a portion of ISCs formed in vivo back to biconcave cells; (3) the
inhibition of ISC formation in vitro is due, in part, to
N-acetylcysteine's ability to block dense cell formation; and (4)
diminution of reduced glutathione (GSH) levels in light density SS
erythrocytes is sufficient to cause dense cell formation. We conclude
that N-acetylcysteine's efficacy in reducing ISC formation is due to
its combined antioxidant activity and ability to increase intracellular
reduced GSH levels.
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MATERIALS AND METHODS |
Preparation of light density and high density SS RBCs.
The methodology used to separate light density and high density SS RBCs
is based on Shartava et al.11 Blood (20 to 30 mL) was
obtained by venipuncture from homozygous SS subjects in vacutainer tubes containing 143 USP units of lithium heparin. Fresh blood (5 mL/gradient) was placed over a discontinuous Percoll density gradient
containing 7 mL each of 45%, 50%, 55%, 60%, and 65% Percoll in
18% Renografin M-60 (Squibb Diagnostics, Princeton, NJ)
in 20 mmol/L HEPES, 10 mmol/L MgCl2, 10 mmol/L glucose, pH
7.4. The blood was sedimented at 907g for 45 minutes at 4°C
and the light density cells (45% layer) were removed without cross
contamination. The low density cells were washed two times in
phosphate-buffered saline (PBS) (10 mmol/L NaPO4, 150 mmol/L NaCl, pH 7.6) and sedimented at 2,520g for 5 minutes. A
2% suspension of low density cells was prepared in incubation buffer
(20 mmol/L HEPES, pH 7.4, 130 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
MgCl2, 1 mmol/L glucose, 2 mmol/L NaPO4, 2 mmol/L CaCl2, 1 mmol/L adenosine, 0.5% bovine serum
albumin [BSA] and 100 U/mL penicillin G, 100 µg/mL streptomycin, 0.25 µg/mL Amphotericin B).
In vitro formation of ISCs by cyclic oxygenation-deoxygenation and
inhibition by reducing agents and antioxidants.
The 2% solution of light density cells in 10 mL of incubation buffer
plus varying concentrations of reducing agents or antioxidants was
placed in 50 mL volumetric flasks. The samples were incubated at
37°C, in a shaker water bath, with cycling of 15 minutes
N2 followed by 5 minutes air for a total time of 16 hours.
After 16 hours, the samples were flushed with O2 for 20 minutes, and aliquots were fixed with 1% glutaraldehyde. Blood smears
were prepared and a minimum of 500 RBCs counted to determine percent ISCs. Cells with a length/width ratio of  2 were counted as ISCs. As
can be seen in Fig 1, cells with multiple
projections were rarely encountered. When they were encountered, they
were not counted as ISCs. In some experiments, cyclic
oxygenation-deoxygenation was followed by density separation as
described above. In these experiments, each layer of the gradient
containing RBCs was washed twice with PBS and the number of RBCs was
counted using the Cell Dyn (Sequoia-Turner, Mountain View,
CA).
Determination of -actin thiols after cyclic
oxygenation-deoxygenation.
For those experiments in which we measured -actin thiols, we began
the cyclic oxygenation-deoxygenation with a 2% solution of light
density cells in 300 mL of incubation buffer. At the end of the 16-hour
cycling, we washed the cells three times with PBS (sedimenting at
2,520g for 5 minutes), isolated ghosts, membrane skeletons, and
-actin according to Shartava et al.11 Thiols were
measured on -actin samples using 5,5 -dithiobis-(2-nitrobenzoate) (DTNB).11
Reduction of intracellular GSH using 1-chloro-2,4-dinitrobenzene
(CDNB).
Low density sickle cells (LDSS) were treated with 1 mmol/L
chloro-dinitrobenzene (CDNB) in incubation buffer at 4°C for
varying times as described in the text. Controls were kept in the same conditions minus CDNB. Incubated LDSS erythrocytes were separated in
Percoll density gradients as described above and GSH per RBC was
measured as follows. RBCs from the Percoll layers were washed with PBS.
Packed RBCs were placed in 25 vol of dH2O for 5 minutes at
22°C. Sufficient 100% trichloro-acetic acid (TCA) was
added to bring the final concentration to 10% TCA, and the mixture was kept on ice for 5 minutes. Precipitate was sedimented (10,000g, 5 minutes). One volume of the supernatant was mixed with 4 vol of 0.5 mol/L Tris pH 8.2. A final concentration of 0.2 mmol/L DTNB (from a 10 mmol/L stock solution in 1% NaHCO3) was added to the
sample and incubated for 30 minutes. After incubation, DTNB absorbance
was read at 412 nm. Molar concentration of GSH was calculated based on
an extinction coefficient E = 13,600 M 1 · cm1.
Conversion of de novo ISCs to the biconcave shape.
High density SS erythrocytes were isolated, from fresh blood, from the
65% Percoll layer upon density separation. The high density SS
erythrocytes were washed three times with PBS and then were brought to
a 2% solution in incubation buffer. The incubation, in the presence of
varying concentrations of N-acetylcysteine (NAC), was performed at
37°C for 16 hours with shaking. At the end of 16 hours, the cells were fixed with glutaraldehyde, blood smears prepared,
and ISCs counted.
Statistical analysis.
Using one-way and two-way analysis of variance (ANOVA), we first tested
for significant differences among the means observed for varying
concentrations of NAC (or dithiothreitol [DTT]). Once the difference was found to be significant, multiple comparisons using
Dunnett's test (for comparing treatments with control) were performed.
The P values were derived through the use of Dunnett's test.
In the matched-paired experiments, we have used two-way ANOVA to take
into account the patient (blocking) effects. In other words, where
appropriate, we have used paired t-test (instead of two-group
t-test) because of its higher power.18,19
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RESULTS |
We first tested the efficacy of a small membrane permeable reducing
agent, DTT, to block the formation of ISCs in vitro
(Table 1). We found that DTT caused
statistically significant decreases in the number of ISCs formed at all
concentrations tested from 1.0 mmol/L to 5.0 mmol/L. At 5.0 mmol/L, we
obtained a reduction of 30.3% in the number of ISCs (Table 1). Higher
concentrations of DTT caused clustering of RBCs.
Next, we wanted to test the efficacy of reducing agents, or
antioxidants, which are in clinical use for other purposes. Cysteamine bitartrate converts cystines to cysteines and cysteine-cysteamine mixed
disulfides. It has been approved by the Food and Drug Administration (FDA) for the treatment of nephropathic cystinosis and
appears to maintain normal renal functions in patients receiving the
drug.20 Cysteamine was ineffective in blocking ISC
formation (at 1 to 50 mmol/L) and actually caused increases in the
number of ISCs at higher concentrations (150 mmol/L and 200 mmol/L)
(data not shown). Succimer (meso 2,3-dimercaptosuccinic acid) and
dimercaprol are used clinically as chelators of heavy metals, such as
lead.21 Succimer was ineffective at blocking ISC formation
in vitro (at 1 to 30 mmol/L) and again caused increases in ISCs at
concentrations of 100 to 200 mmol/L (data not shown). Dimercaprol
caused a statistically significant decrease in ISCs at 10 µm
concentration ( 20.5%), but at higher concentrations
(0.1 to 1.0 mmol/L) caused increases in percent ISCs (data not shown).
NAC is an antioxidant and is converted intracellularly to L-cysteine,
which is a precursor to reduced glutathione. We felt that the combined
activities of this drug might make it particularly efficacious in
blocking ISC formation in vitro. As shown in
Table 2, NAC caused statistically
significant decreases in percent ISCs at concentrations of 250 µmol/L
to 20 mmol/L NAC. At 20 mmol/L NAC, there was a reduction of 35.7%
ISCs. NAC (20 mmol/L) had no effect on cell lysis during the 16 hours
of cycling, as evidenced by identical concentrations of hemoglobin
(measured by absorbance at 430 nm) in the incubation mixtures ± NAC
(data not shown). Figure 1 shows a typical smear of ISCs formed in
vitro in the presence (Fig 1C) or absence (Fig 1B) of 20 mmol/L NAC.
To test whether NAC might affect SS erythrocyte dehydration, we
isolated light density SS RBCs and incubated them at 37°C under
cyclic deoxygenation-reoxygenation for 16 hours plus
(Fig 2C) or minus (Fig 2B) 20 mmol/L NAC.
The control represents LDSS erythrocytes, which were incubated at
37°C for 16 hours minus NAC and without cycling (Fig
2A). As shown in Fig 2 and Table 3, in
vitro cycling of LDSS erythrocytes causes a large conversion to high
density SS erythrocytes. NAC (20 mmol/L) partially blocks this
conversion causing statistically significant decreases in the percent
RBCs found in the 60% Percoll layer (density = 1.112 g/mL) and
increases in the percent RBCs found at lower density (45% and 50%
Percoll layers, density = 1.093 and 1.100 g/mL) (Table 3). There was no
statistically significant difference in the percent ISCs found at any
Percoll layer ± 20 mmol/L NAC. This means that decreases in ISCs
found in whole blood smears from NAC-treated cycled cells (Table 2) was
accompanied by NAC blocking the formation of high density SS cells in
vitro (Table 3), which contain the highest percent ISCs.
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| Fig 2.
Effect of NAC on SS erythrocyte density. Low density SS
erythrocytes that were incubated at 37°C for 16 hours without
cycling or NAC (A), with cycling, but without NAC (B), and with cycling in the presence of 20 mmol/L NAC (C), were placed on Percoll gradients (45% to 65%) and centrifuged at 907g for 45 minutes. Note
NAC's ability to inhibit dense cell formation. Density of the RBCs in different Percoll layers was obtained by sedimenting density marker beads (Pharmacia, Piscataway, NJ) under identical
conditions.
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We have previously shown that light density ISCs, formed by in vitro
cycling in the presence of NO3,
demonstrate slow dissociation of the spectrin membrane
skeleton.22 This result uncoupled the ISC membrane skeleton
defect from the hydration status of the SS erythrocyte. Therefore, we
wanted to ask whether NAC might also be having a direct effect on SS
erythrocyte -actin. To determine whether -actin thiols might be
one target of NAC, we repeated the NAC experiments with larger amounts
of light density SS RBCs as our starting material (300 mL of a 2% RBC
solution). This allowed us sufficient material to count the number of
ISCs after 16 hours of cycling, as well as measure the number of
-actin thiols with DTNB.11 In four independent
experiments, we found that 20 mmol/L NAC caused a statistically
significant decrease of 32.8% ± 5.7% ISCs as compared with
samples that were cycled in the absence of NAC. There was an inverse
trend showing increases in -actin thiols in the presence of 20 mmol/L NAC. These same 20 mmol/L NAC samples were found to have a
statistically significant increase of 23.1% ± 8.2% available
-actin thiols (Table 4). The available
thiols were increased from 1.3 ± 0.1 in the absence of NAC to 1.6 ± 0.2 in the presence of 20 mmol/L NAC. An increase of 0.3 thiols
is consistent with  ISCs being 32.8%. We concluded that NAC does
effect the number of available thiols in sickle cell -actin. The
lack of correlation, from experiment to experiment, in the magnitude of
ISC reduction and -actin thiol increase could be taken to mean that
actin thiol status and ISC morphology are mechanistically unrelated.
But this is not a likely explanation given our previously published
results.11-15 A more likely explanation for the lack of
correlation is the small number of experiments along with high
patient-to-patient variability.
It was clear that NAC had the ability to block the formation of dense
cells and ISCs in vitro and that it had at least two targets: channels
involved in cell dehydration and membrane skeletal -actin. We next
wanted to test whether NAC could convert ISCs back to biconcave cells.
To answer this question, we isolated dense cells from six independent
homozygous SS subjects. In six separate experiments, with six different
sickle cell patients, the dense cell population contained a mean of
61.2% ± 5.1% de novo ISCs. Dense cells obtained from the 65%
Percoll layer were incubated for 16 hours at 37°C in our incubation
buffer ± 20 mmol/L NAC. Those dense cells incubated in the absence
of NAC contained 49.7% ± 4.9% ISCs indicating that the incubation
buffer alone can cause a significant reduction in ISCs during a 16-hour
incubation at 37°C. Those incubated with 20 mmol/L NAC contained
42.2% ± 3.8% ISCs, which represented a statistically significant
mean percent difference of 14.0% ± 4.1% (P < .05).
Therefore, NAC has the ability to convert de novo ISCs back to the
biconcave shape.
Due to the increased deposits of heme and free iron on the cytoplasmic
surface of the high density SS erythrocyte membrane and the resultant
formation of superoxide, peroxides, and hydroxyl radicals,6,23,24 we have proposed that the diminished
levels of GSH in these same dense cells25,26 is critical to
oxidative damage to -actin and other target proteins.15
We further proposed that this oxidative damage to -actin, and other
targets, leads to the formation of dense cells and ISCs. To test this
hypothesis, we incubated low density sickle cells at 4°C in the
presence and absence of 1.0 mmol/L CDNB. CDNB lowers GSH levels within
erythrocytes by forming an irreversible adduct,
2,4-dinitrophenol-S-glutathione.27 As shown in the kinetic
experiments presented in Fig 3, there is a
rapid decrease in GSH levels even after 1 hour of incubation with CDNB,
reaching a 90% decrease after 24 hours. The percentage of high density
cells (those found in the 60% and 65% Percoll layers) increases
constantly at 1 hour, 12 hours, and 24 hours CDNB treatment. Therefore,
diminishing GSH levels in CDNB-treated cells is sufficient to cause
cellular dehydration.
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| Fig 3.
Diminishment of GSH leads to formation of dense cells.
Percent of high density cells (% HDSS) formed were calculated and
plotted after density separation via Percoll gradients of SS RBCs,
which underwent incubation at 4°C of low density (45% Percoll
layer) sickle cells without ( ) or with ( )
1 mmol/L CDNB. Mol concentrations of GSH were determined in the same
RBCs in the absence ( ) and in the presence
( ) of 1 mmol/L CDNB. Data is presented as mean ± standard error of four independent experiments. Values for GSH and HDSS
were statistically significant (P < .05) at 1 hour, 12 hours,
and 24 hours when comparing ± CDNB samples.
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| |
DISCUSSION |
Hebbel et al23 have shown that sickle cells generate about
twice the amount of activated oxygen species found in control RBCs. The
reason for this increase in oxygen radicals is the result of
accelerated autoxidation of HbS to methemoglobin, a conversion that
causes release of heme.6 Heme is increased in content on
the cytoplasmic surface of sickle cell membranes and this correlates with membrane thiol modification.24 The amount of heme and
free iron associated with the cytoplasmic surface of the SS erythrocyte membrane is particularly increased in the most dense fractions of SS
RBCs.6 Reduced glutathione levels are diminished by about 20% in SS RBCs as compared with high reticulocyte controls and is
lower in high density SS erythrocytes compared with low density SS
erythrocytes.25,26 The diminished levels of reduced
glutathione are related to decreased glutathione reductase activity,
increased glutathione peroxidase activity, and inhibition of the
pentose phosphate shunt in SS erythrocytes.25 Therefore,
sickle cells have increased activated oxygen species, and dense cells
also have diminished levels of GSH to protect against oxidative damage. This combination is probably responsible for oxidation of thiol groups
in many target proteins.
In this study, we show that an antioxidant, NAC, can block dense cell
formation in vitro. Dense cell formation is caused by water loss from
sickle cells, in response to K+ leakage. The cation
transport systems that appear to be involved in dehydration of SS
erythrocytes are the K+-Cl
cotransporter, the Ca2+-activated K+ channel,
and the deoxygenation-induced Na+, K+, and
Ca2+ leak pathways.8 Therefore, these are the
most likely targets of NAC protection of oxidative damage leading to
decreased dense cell formation. Because the dense cells contain the
highest percent ISCs, this supplies a partial explanation of NAC's
effect on ISC formation. But it does not get directly at the molecular
mechanism of NAC's effect on ISC formation. Dense cells have decreased
fetal hemoglobin and increased hemoglobin S concentration leading to greater polymerization of HbS.6,7 This would cause many of the high density SS erythrocytes to become sickled in shape. But what
causes the cell to lock into an irreversibly sickled shape? This cannot
be explained by dehydration alone for several reasons. First, some ISCs
in blood from SS subjects are low density cells.6-8 Furthermore, we have shown that substitution of
NO3 for Cl in the in
vitro deoxygenation-reoxygenation cycling experiments results in a high
concentration of low density ISCs.22 These low density ISCs
show slow dissociation of their membrane skeleton at
37°C.22 Therefore, the formation of ISCs can be
uncoupled from cellular dehydration of SS erythrocytes. While dense
cell fractions contain many sickled cells, we believe that the locking of the ISC requires a modification in the membrane
skeleton.15
The C284-C373 disulfide bridge in -actin is the key determinant of
the slow dissociation of the ISC membrane skeletal components and
therefore the inability of the ISC to remodel its
shape.11-15 We therefore proposed that reducing agents and
antioxidants might be effective in blocking ISC formation in vitro. Of
the clinically relevant antioxidants that we have tested, NAC was the
most efficacious in inhibiting ISC formation in vitro. Furthermore, the
NAC-linked reduction in ISCs was accompanied by an increase in
-actin thiols. Therefore, in this study we have identified two
possible targets for NACs blockage of dense cell and ISC formation:
channels involved in K+ leakage and -actin.
NAC, as the N-acetyl derivative of L-cysteine, is an
antioxidant.28 It is highly permeable to cell membranes,
and within the cytoplasm, is converted to L-cysteine, which is a
precursor to GSH.29 It, therefore, could protect thiols
from oxidative damage both by its antioxidant capacity, as well as
raising the levels of GSH. Indeed, adult respiratory distress syndrome
(ARDS) patients treated with NAC (70 mg/kg) show a 30%
increase in erythrocyte glutathione in 1 day and a 47% increase after
4 days with drug administered every 8 hours.30 In this
regard, our experiments showing that lowering GSH levels (with CDNB)
can lead to dense cell formation are of substantial interest. In the
future, we must determine whether replenishment of GSH with NAC can
reverse the effect of CDNB on dense cell formation in vitro.
| |
FOOTNOTES |
Submitted March 5, 1997;
accepted January 23, 1998.
Supported by Grant No. 3P60 HL38639 from the National Institutes of
Health, Bethesda, MD, to the USA Comprehensive Sickle Cell
Center. S.R.G. serves as Principal Investigator and Program Director.
Address reprint requests to Steven R. Goodman, PhD,
Professor and Chairman, Department of Structural and Cellular Biology, University of South Alabama College of Medicine, MSB 2042, Mobile, AL
36688.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Kathy Billingsley and Ashley W. Turbeville for
manuscript preparation.
| |
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Abstract
Introduction
Abstract
