Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 376-384
Evaluation of Biochemical Changes During In Vivo Erythrocyte
Senescence in the Dog
By
Michael P. Rettig,
Philip S. Low,
J. Aura Gimm,
Narla Mohandas,
Jiazhen Wang, and
John A. Christian
From the Departments of Chemistry and Veterinary Pathobiology, Purdue
University, West Lafayette, IN; and Lawrence Berkeley National
Laboratory and UCSF/UCB Bioengineering Graduate Group, Berkeley, CA.
 |
ABSTRACT |
One hypothesis to explain the age-dependent clearance of red blood
cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn,
band 3 clustering generates an epitope on the senescent cell surface
leading to autologous IgG binding and consequent phagocytosis. Because
dog RBCs have survival characteristics that closely resemble those of
human RBCs (ie, low random RBC loss, 
115-day life span), we decided
to test several aspects of the above hypothesis in the canine model,
where in vivo aged cells of defined age could be evaluated for
biochemical changes. For this purpose, dog RBCs were biotinylated in
vivo and retrieved for biochemical analysis at various later dates
using avidin-coated magnetic beads. Consistent with the above
hypothesis, senescent dog RBCs were found to contain measurably
elevated membrane-bound (denatured) globin and a sevenfold enhancement
of surface-associated autologous IgG. Interestingly, dog RBCs that were
allowed to senesce for 115 days in vivo also suffered from compromised
intracellular reducing power, containing only 30% of the reduced
glutathione found in unfractionated cells. Although the small quantity
of cells of age 
110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster
in response to hemichrome binding. Importantly, band 3 in RBCs 112
days old was found to be 25% less restrained by skeletal interactions
than band 3 in control cells, indicating that the normal linkages
between band 3 and the membrane skeleton had been substantially
disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly
assumed to reflect RBC age, was found to be maximal in RBCs isolated
only 58 days after labeling, implying that while this marker is useful
for identifying very young populations of RBCs, it is not a very
sensitive marker for canine senescent RBCs. Taken together, these data
argue that several of the readily testable elements of the above
hypothesis implicating band 3 in human RBC senescence can be validated
in an appropriate canine model.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN red blood cells (RBCs) have a
circulating life span of 120 days, after which they are recognized
and removed from circulation by macrophages of the mononuclear
phagocyte system.1 Although characterization of the
mechanisms that permit selective phagocytosis of senescent cells has
been undertaken by many,2 progress has been hampered by the
lack of a model system that allows isolation of truly senescent cells
after their aging in vivo.2 Although density gradient
separation methods have allowed the collection of human RBCs that are
destined for immediate removal,3 there is still concern
that the densest cells may not be truly senescent.4-6
Development of hypertransfusion7 and biotinylation methodologies8 to isolate populations of unquestionably
senescent RBCs from live animals has successfully generated much
information on changes that occur during RBC
senescence,4-7,9-17 but these studies may not be relevant
to human RBCs because they have used animals (rabbit, rodent) that
exhibit high random (age independent) RBC removal and short RBC life
spans (50 to 70 days).18,19 To circumvent this problem,
we have adapted the biotinylation method for application in the
dog,20 largely because dog RBC survival characteristics are
very similar to human RBCs in exhibiting low random
removal18 and a long life span of 115
days.18,21,22
Because mature RBCs are unable to synthesize new proteins, the events
that trigger removal of senescent cells from circulation must derive
from alterations in pre-existing proteins or lipids that lead to
recognizable changes at the membrane surface. We and others have
proposed that one such alteration is the age-dependent clustering of
the membrane-spanning protein, band 3.23-29 According to
this hypothesis, band 3 clustering can arise from multiple mechanisms
that are active late in an RBC's life span, including hemoglobin (Hb)
denaturation23,25 and protein
oxidation.26,27,30 Upon formation of band 3 clusters, there
is considerable evidence for an obligatory opsonization of the clusters
by autologous antibodies.24,26,27,31-34 Evidence to date
suggests that these autologous anti-band 3 antibodies consist
predominantly of IgG subclass 2 and 3.31 However, the exact
antigenic determinate on band 3 is controversial because both
carbohydrate26,35 and protein36 epitopes on the
anion transporter have been reported. In addition, anti-band 3 antibodies are able to bind complement component C337 and
form C3b-IgG complexes.38 These C3b-IgG complexes can
nucleate alternative complement pathway C3 convertases resulting in the
deposition of additional complement components on the senescent cell
surface.39 Eventually, the RBC becomes coated with
sufficient antibody and complement to be recognized and removed from
circulation by macrophages of the mononuclear phagocyte
system.1,2,24,27,40
The pathway of RBC removal discussed above has been primarily
characterized by studying pathologic (sickle and
thalassemic),32,34 density separated,33,38 or
in vitro-modified RBCs.24,26,27 However, very little
evidence supporting the pathway has been obtained from normal in
vivo-aged RBCs that are unquestionably senescent and have survival
characteristics similar to humans. Our preliminary studies using the
biotinylation system in the dog have shown that dog RBCs greater than
104 days old bind significantly more autologous IgG than a random aged
population of dog RBCs.41 In this report, we have examined
several additional predictions of the clustering hypothesis of RBC
senescence in the dog.
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MATERIALS AND METHODS |
In vivo biotinylation of canine RBCs.
Two dogs (nos. 1630 and 5005) used in these studies were healthy,
mature male beagles obtained from a commercial random source vendor.
They were housed indoors and maintained according to Purdue University
Animal Care and Use Committee regulations. Both dogs were considered
healthy as determined by hematology and clinical biochemistry profiles
and by parasite (intestinal, blood) testing.
To produce a larger population of biotinylated RBCs for collection at
the end of the RBC life span, biotinylation was performed during
reticulocytosis in response to an iatrogenic blood loss anemia. Dog no.
5005 was bled three times over a 3-day period, reducing the hematocrit
(Hct) to 30% (prebleeding Hct, 49%), and biotinylation was performed
7 days after the first bleeding. Dog no. 1630 was bled three times over
a 4-day period, reducing the Hct to 33% (prebleeding Hct, 50%), and
biotinylation was performed 13 days after the first bleeding.
Both dogs had 97% to 100% of circulating RBCs biotinylated by
intravenous (IV) infusion of N-hydroxysuccinimide biotin
(NHS-biotin; Fluka, Milwaukee, WI) dissolved in dimethyl sulfoxide, as
described previously.20 Briefly, NHS-biotin was dissolved
in medical grade 90% dimethyl sulfoxide (DMSO; Syntex, West Des
Moines, IA) and administered very slowly through an indwelling (IV)
catheter. Both dogs initially received 35 mg of NHS-biotin per kg body
weight over approximately 30 minutes. However, because of a low
concentration of biotin per RBC for dog no. 5005, an additional 17 mg
NHS-biotin per kg body weight was administered 4 days after the initial
biotinylation. Pretreatment of both dogs with atropine sulfate (0.05 mg/kg) was used to counteract cholinergic-like side effects of the
DMSO.
Isolation of biotinylated RBCs.
Detailed procedures for the isolation of in vivo-aged biotinylated
RBCs by magnetic bead activated cell sorting (MACS) have been described
elsewhere.20 Briefly, heparinized whole blood was washed
three times in cold PBS-G (125 mmol/L sodium chloride, 20 mmol/L sodium
phosphate, pH 7.4, 5 mmol/L EDTA, 0.01% sodium azide, 5 mmol/L
glucose), removing the buffy coat and plasma proteins. The percentage
of biotinylated RBCs in circulation was determined by incubating an
aliquot of RBCs (2% Hct) with avidin-fluorescein isothiocyanate
(avidin-FITC) (0.033 mg/mL; Sigma, St Louis, MO, or Becton Dickinson,
San Jose, CA) at 37°C for 30 minutes. RBCs were then washed three
times with PBS-G and resuspended in BSA solution (1% bovine serum
albumin in PBS-G) for analysis using a Coulter Epic Elite series flow
cytometer (Coulter, Hialeah, FL). Biotinylated cells in
washed, unfractionated samples were labeled for sorting by incubating
with avidin-FITC in PBS-G for 10 minutes on ice (0.188 mg avidin-FITC
per 0.150 mL packed biotinylated RBCs). The cells were washed twice in
PBS-G followed by a 5-minute incubation on ice with biotinylated MACS
microbeads (0.0676 mL microbeads per 0.150 mL packed biotinylated RBCs;
Miltenyi Biotec, Auburn, CA). In addition to serving as a cross-linker,
the avidin-FITC served as an internal fluorescent marker for assessment
of purification efficiency.
For sorting, the RBCs were diluted to 2% hematocrit in BSA solution,
applied to an MACS separation column (size "D" from Miltenyi Biotec) containing plastic coated steel wool, and placed in the magnetic field of a permanent magnet (providing 0.6 T; obtained locally). The biotinylated RBCs (positive fraction) adhered to the
steel wool and nonlabeled RBCs (negative fraction) were eluted with BSA
solution. Nonretrieved RBC were diluted to 1% Hct with BSA solution,
reapplied to the column in the presence of the magnet, and eluted with
BSA solution. The column was then extensively washed with BSA solution,
removed from the magnet, and biotinylated RBCs were flushed from the
column.
Quantitation of intracellular reduced glutathione (GSH).
Reduced glutathione was determined as described by Beutler et
al.42 The method was adapted for samples containing 20 µL of 50% Hct RBCs.
Analysis of RBC membranes by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and immunoblotting.
RBC ghost membranes were prepared from isolated cells by hypotonic
lysis in 5 mmol/L sodium phosphate, 1 mmol/L EDTA, pH 8.0 in the
presence of 40 µg/mL phenylmethyl sulfonyl fluoride (lysis buffer)
according to the procedure of Dodge et al.43 The lysate was
centrifuged at 12,000g for 40 minutes and the supernatant was
removed. The membrane pellets were resuspended and washed three
additional times in lysis buffer. Protein content of the membranes was
determined by the bicinchoninic acid method (BCA; Pierce, Rockford, IL)
and membranes were solubilized in SDS electrophoresis buffer containing
100 mmol/L dithiothreitol and heated 3 minutes in a boiling water bath.
For dog no. 5005, an aliquot of each membrane sample was stored
overnight at 4°C for analysis by gel electrophoresis the following
day. The remaining ghost membranes from dog no. 5005 and all of the
membranes from dog no. 1630 were stored at 
80°C until
completion of the final isolation. Ghost membranes were resolved on 6%
to 12% gradient polyacrylamide gels and were either stained for
protein with Coomassie brilliant blue (Sigma) or transferred to
nitrocellulose (pore size 0.2 µm; Schleicher and Schuell, Keene, NH)
using the buffer system of Towbin et al44 and blocked with
5% milk in TBS-T (20 mmol/L Tris, pH 7.5, 500 mmol/L NaCl, 0.05%
Tween 20). To develop, the immunoblot was washed with TBS-T and
incubated at room temperature for 3 hours in a solution of rabbit
anti-dog Hb (Research Plus, Bayonne, NJ) diluted 1/5,000 in TBS-T with
1% milk. After further washing and incubation with peroxidase-labeled
goat anti-rabbit IgG (Bio-Rad, Hercules, CA), the immunoblots were
developed using an enhanced chemiluminescence (ECL) reagent kit
(Amersham, Arlington Heights, IL) followed by exposure to film. To
determine if the banding patterns in the SDS-polyacrylamide gels of our
ghost membranes were altered during storage at 80°C, an
aliquot of each sample from dog no. 5005 was resolved the day
immediately after preparation. We found no observable differences
between the banding patterns in samples analyzed immediately after
preparation and the samples stored at 80°C (results not
shown).
Fluorescence-imaged microdeformation assay of dog RBCs.
Dog RBCs were washed two to three times with PBS (PGS-G with no
glucose) containing 0.01% BSA (labeling buffer), and band 3 was
labeled in situ with eosin-5-maleimide (Molecular Probes, Eugene, OR).
The washed RBCs were incubated at room temperature for 45 minutes in
100 µg/mL of eosin-5-maleimide in labeling buffer and then washed
three times with labeling buffer. Association of band 3 with the
membrane skeletal network through ankyrin was determined by measuring
its redistribution in response to mechanical deformation.45,46 Individual cells were partially aspirated into a glass micropipette and a fluorescent image was taken with a
liquid nitrogen cooled charge-couple device (CCD) camera (Photometrics, Tucson, AZ). The fluorescence intensity provides a measure of the
density of the labeled band 3 along the membrane protrusion extending
into the micropipette, and the intensity changes along the aspirated
length provide information on the mobility of band 3 relative to the
underlying spectrin/actin skeleton.
Iodination of protein A.
Standard procedures were used to radiolabel protein A (Pierce) with
125iodine (125I; NEN Life Science Products,
Boston, MA) using Iodobeads (Pierce).47 Iodobeads were
washed thoroughly in PBS and dried on filter paper before use. To three
Iodobeads in a 1.5-mL centrifuge tube was added 0.5 mL of PBS and 0.3 mCi 125I. The mixture was allowed to sit at room
temperature for 5 minutes, after which 250 µg of protein A in 0.5 mL
of PBS was added. Following incubation at room temperature for 30 minutes, the unreacted free 125I was removed by passage
through a desalting column (10DG from Bio-Rad). Protein concentration
was determined by the BCA method and radioactivity was measured in a
Packard Cobra Gamma counter (Packard Instrument Co, Meriden, CT).
Radiolabeled protein A was stored at 4°C and used within 1 month.
125I protein A binding assay: Detection of autologous
IgG binding to dog RBCs.
Isolated biotinylated (positive fraction) and non-biotin-labeled
(negative fraction) RBCs were suspended at 40% Hct in PBS containing
4% BSA. To determine autologous IgG binding, 25-µL aliquots of the
positive and negative fractions were mixed with 0.2 µg of
125I-protein A and incubated for 30 minutes at room
temperature with gentle shaking. The mixtures were washed five times in
PBS buffer containing 2% BSA to remove unbound radiolabel, transferred
to new tubes, and counted in a Packard Cobra Gamma counter. The number of RBCs per sample was determined by manual RBC counts (3 replicates per aliquot). The number of protein A molecules per RBC was calculated from these data.
| |
RESULTS |
Biotinylation, survival, and magnetic cell sorting.
IV infusion of NHS-biotin labeled greater than 97% of circulating dog
RBCs. Both dogs (nos. 1630 and 5005) exhibited normal RBC life span
based on previous labeling studies (data not shown).20 During the current study, blood was collected from the dogs, and biotinylated (positive) RBCs were isolated by magnetic cell sorting. Nonbiotinylated (negative) RBCs were also collected and used as control
cells. In some assays, a presorted RBC sample containing a mixture of
positive and negative cells was collected to serve as an additional
control. The percentage of biotinylated RBCs in the isolated positive
fraction after magnetic cell sorting was always greater than 72% and
averaged 88%.
Intracellular reduced GSH in dog senescent RBCs.
Although the band 3 clustering hypothesis of senescent RBC clearance
predicts that senescent cell phagocytosis is triggered by antibody
binding to hemichrome-induced aggregates of band 3, little effort has
been devoted to identifying the cause of hemichrome formation from
oxidized/denatured Hb in late-term erythrocytes. Believing that this
latter process might derive from an inability to maintain an adequate
reducing power in the cell,2,25,26 we examined the reduced
GSH content of the in vivo-aged canine RBCs as a function of cell age.
Cellular GSH levels were assayed in control (both presort and negative)
and positive (biotinylated) RBC fractions isolated from dog no. 1630 on
3 separate days near the end of the biotinylated RBC's life span. As
shown in Fig 1, unfractionated erythrocytes
(solid dots), RBCs not retrieved with avidin-coated beads at day 108 (<108 days old, open circles) and biotinylated RBCs collected on the
same day (108 days old, solid triangles) all contained 10 to 12 µmol GSH/g Hb in their cells. However, over the next 7 days, as the
biotinylated cells reached an age of at least 115 days, their GSH
content declined to 1/3 of their value a week earlier, while the
nonretrieved cells (<115 days old) as well as the unfractionated
cells retained their high (12 µmol/g Hb) GSH content. These data
suggest that a precipitous change in intracellular reducing power
occurs near the end of the RBC's life span. Although the limited
quantities of late-term biotinylated cells prohibited collecting data
from multiple dogs, the observation that neither the unfractionated
RBCs nor the fractionated RBCs that failed to bind beads (younger
cells) from the same dog displayed any decrease in cellular GSH levels
suggests that the changes observed in the biotinylated cells are real.
However, whether this behavior will prove to be a characteristic of RBC senescence in all dogs will obviously require further scrutiny.
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| Fig 1.
Intracellular reduced glutathione concentrations of in
vivo-aged biotinylated dog RBCs. Positive ( ) biotinylated and
negative ( ) RBCs were isolated from dog no. 1630 at the indicated
times post-biotinylation by magnetic cell sorting. In addition, a
presort ( ) RBC sample was collected from dog no. 1630 before each
separation by magnetic cell sorting. Samples were assayed in triplicate
for intracellular reduced glutathione and the mean (±SD) is shown.
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Analysis of membrane associated Hb.
For Hb denaturation to participate in triggering the removal of a
senescent erythrocyte from circulation, denaturation/oxidation must be
minimal in young and middle-aged erythrocytes, but prominent in cells
near the ends of their life span. To determine when Hb denaturation
occurs during a RBC's life span in vivo, membranes were isolated from
RBCs retrieved at several time points after biotinylation, separated
electrophoretically by polyacrylamide gel electrophoresis, and
immunoblotted with an antibody specific for dog Hb. As shown in the
immunoblot of Fig 2A, a significant increase in the quantity of membrane-bound globin appears on the membranes of cells isolated 86 days after biotinylation of dog no. 5005 (mean cell age 
100 days). A similar increase in membrane-bound globin
appeared on the membranes of cells isolated from dog no. 1630 (data not
shown). The binding of small quantities of Hb to dog membranes prepared
from a random aged cell population (lane day 1) was expected and has
been shown to also occur in freshly prepared human
membranes.48,49 Interestingly, the amount of globin monomer
in the immunoblot does not change significantly with cell age. However,
the quantity of covalently cross-linked forms of globin are found to
increase dramatically as the cell approaches the end of its life (Fig
2B), demonstrating that defects in Hb maintenance begin to emerge late
in an RBC's life span.
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| Fig 2.
Western blot analysis of globin deposition on the
membranes of in vivo-aged biotinylated dog RBCs. Positive
(biotinylated) RBCs were isolated from dog no. 5005 at the indicated
times post-biotinylation by magnetic cell sorting. (A) Ghost membranes
were prepared as described in the Materials and Methods. At the end of
the study, membranes (20 µg) were electrophoresed, transferred to
nitrocellulose, and analyzed for globin by immunoblotting as described
in Materials and Methods. The lane marked Hb contains 5 µg of soluble
Hb obtained from a dog RBC lysate. (B) Results of scanning densitometry
of the dimeric globin band seen in the immunoblot in (A). The
integrated area of the globin dimer band is plotted as a function of
the minimum age of the biotinylated cells. A similar increase in
membrane-bound globin appeared on the membranes of cells isolated from
dog no. 1630 (data not shown).
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Analysis of band 3 during RBC senescence in vivo.
During the course of these studies, we attempted to examine the
aggregation state of band 3 in C12E8 extracted
dog ghost membranes by size-exclusion high-performance liquid
chromatography.50 However, we found it very difficult to
extract the spectrin and actin from dog erythrocyte membranes in
preparation for analysis of the cluster size of band 3. It was also
problematic to isolate the quantities of senescent cells necessary for
the above analyses. Consequently, we elected to look for changes in the
properties of band 3 using a methodology that could be readily
conducted on small quantities of whole cells.
The technique of fluorescence-imaged microdeformation (FIMD) was
originally developed to evaluate the distribution of labeled molecular
components in a biological membrane during a mechanically induced
deformation of that membrane.45,46 Using this technique, Discher et al45 showed that band 3, which is normally
30% associated with the membrane skeleton,50,51
exhibits a concentration gradient upon deformation that is intermediate
between free-flowing lipid and skeletally attached proteins. In the
present study, we used FIMD to evaluate the distribution pattern of
labeled band 3 in the dog erythrocyte as a function of cell age.
Positive (biotinylated) and negative RBCs isolated by magnetic cell
sorting were fluorescently labeled with eosin maleimide (specific for
band 3) and aspirated into a micropipette. Fluorescence images of
deformed cells were collected and analyzed.
Figure 3 illustrates the technique used and
the results obtained with the positive and the negative cells collected
from dog no. 1630 on day 112 post-biotinylation. As shown in Fig 3,
entrance density of band 3 (
e) increases and the cap
density of band 3 (c) decreases with increasing
projection length (L/Rp). However, the extent of increase
in e and the extent of decrease in c was
lower for the positive cells compared with the negative cells. This
finding is illustrated in the plot of e c versus L/Rp. The positive cells had a
linear slope of 0.114, while the negative cells gave a steeper slope of
0.150. Similar changes were also noted for positive and negative cells isolated at days 93 and 107 post-biotinylation. Because the slope of
this plot is a measure of the "cytoconnectivity" of band 3, it
can be concluded that there is a 25% reduction in the number of band 3 molecules attached to the underlying membrane skeleton in senescent dog
RBCs compared with the randomly aged RBCs. Importantly, no differences
in e and c values were noted between
positive and negative cells isolated at day 58 post-biotinylation.
Although the general applicability of these findings to other dogs and humans will require further analysis, the existing data nevertheless imply that band 3 mobility increases at the end of RBC life span.
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| Fig 3.
Fluorescence-imaged microdeformation (A through C) and
the comparison of the difference in entrance and cap density for band 3 in positive and negative dog RBCs (D through F). (A through C) A bright
field image of a micropipette-aspirated RBC (A), equivalent fluorescent
image (B), and integrated fluorescence-density profile (C) used for the
analysis. The collected density profiles from the negative (D) and
positive RBCs (E) at 112 days post-biotinylation are plotted in (D) and
(E), respectively. The composite data of
e-c against L/Rp for positive
and negative cells are shown in (F).
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Detection of autologous IgG on in vivo-aged dog RBCs.
Although direct clustering of band 3 in senescent canine erythrocytes
could not be evaluated, microdeformation assays showed that impediments
to band 3 aggregation disappear near the end of the cell's life span.
Because numerous model studies have shown that hemichromes aggressively
cluster mobile band 3 and that these clusters bind autologous
IgG,52 we decided to evaluate the time course of autologous
IgG binding during RBC senescence.
The amount of cell-surface-bound IgG on dog RBCs collected on various
days post-biotinylation by magnetic cell sorting was estimated by a
125I-protein A binding assay
(Fig 4). Each erythrocyte sample was assayed in triplicate on the day the RBCs were isolated, and the displayed data represent the average of the triplicate samples from two
dogs. The graph shows that the amount of autologous IgG bound to
senescent dog biotinylated RBCs increases dramatically only at the end
of the RBC's life span. By assuming that the binding ratio for protein
A to IgG is approximately one to one,53,54 positive
(biotinylated) senescent dog RBCs can be calculated to bind 150
molecules of IgG at the end of their life span, while negative
(control) RBCs can be shown to contain only 20 molecules of IgG over
the remainder of their life in circulation.
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| Fig 4.
IgG binding to in vivo-aged biotinylated dog RBCs.
Positive () and negative () RBCs were isolated at the indicated
times post-biotinylation by magnetic cell sorting. Isolated RBCs were
incubated with 125I protein A and then washed five times
before determining the number of protein A molecules per RBC. Each
fraction was assayed in triplicate, and each point represents the mean ± 1 SD of two dogs. Day 0 data was obtained using RBCs collected just
before the in vivo biotinylation.
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Conversion of protein 4.1b to 4.1a during the dog RBC life span.
As with human protein 4.1, dog protein 4.1 is converted from 4.1b to
4.1a by the deamidation of asparagine 502 in a time-dependent manner.55 Interestingly, this simple conversion of
asparagine 502 to aspartic acid is accompanied by an increase in the
apparent molecular mass of 4.1b (78 kD) to that of 4.1a
(80 kD) on SDS-polyacrylamide gels. Figure
5 shows the pattern of dog RBC membrane proteins band 3, 4.1, and 4.2 on Coomassie brilliant blue-stained SDS-polyacrylamide gels as a
function of cell age. Control membranes, prepared from cells obtained
before biotinylation, represent a random aged cell population (mean
cell age, 55 days) and contain approximately 60% 4.1a and 40% 4.1b
(Fig 5, control lanes). Figure 5A shows the banding pattern of protein
4.1 species in cells that did not associate with the avidin-coated
beads (negative fraction). Because 97% of all erythrocytes were
initially biotinylated, these negatively sorted cells are assumed to be
no older than the day on which they were isolated. Furthermore, their
average age should be only half of this "isolation" age, because
new nonbiotinylated cells will have been added to the population daily.
As expected, protein 4.1 in the negative RBC fraction was initially
enriched in 4.1b (day 23 sample; mean cell age, 12 days) and with
time, exhibited protein 4.1a and 4.1b concentrations that are
representative of a random aged cell population (Fig 5A, day 58 to
112). Figure 5B and C show the conversion of 4.1b to 4.1a in
biotinylated (positive) cells as a function of increasing cell age.
Surprisingly, there was nearly complete conversion of 4.1b to 4.1a in
cells isolated on day 58 post-biotinylation for both dog no. 1630 (Fig
5B) and dog no. 5005 (Fig 5C). This deamidation rate for dog protein
4.1b would appear to be significantly faster than what has been
previously reported for human protein 4.1 (see
Discussion).56
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| Fig 5.
SDS-PAGE analysis of protein 4.1 in age-defined dog RBC
membranes. RBCs were isolated from dog no. 1630 or dog no. 5005 at the
indicated times post-biotinylation by magnetic cell sorting and ghost
membranes were prepared as described in Materials and Methods.
Equivalent amounts (20 µg) of membrane protein were electrophoresed
on gradient (6% to 12%) polyacrylamide gels and stained for protein
with Coomassie brilliant blue. (A) Membranes from nonbiotinylated cells
retrieved from dog no. 1630; (B) membranes from biotinylated cells
retrieved from dog no. 1630; (C) membranes from biotinylated cells
retrieved from dog no. 5005. C denotes control cells not subjected to
biotin-avidin sorting.
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| |
DISCUSSION |
We and others have proposed a mechanism of senescent cell recognition
and removal that depends on the redistribution of the membrane-spanning
protein, band 3.23-29 Specifically, we have shown that the
oxidative denaturation of Hb leads to formation of band 3 clusters due
to the avid association between hemichromes and the cytoplasmic domain
of band 3.23,57-59 These band 3 clusters on senescent cells
are recognized by the immune system as foreign and become opsonized
with autologous IgG and complement.24,26,27,31-34 Upon
deposition of sufficient antibodies and complement, the senescent cell
is then recognized by macrophages and removed from circulation by
phagocytosis.1,39,40 In this report, we have examined this
mechanism of RBC removal for the first time using cells of known age
that were allowed to senesce by natural mechanisms in vivo.
The globin immunoblot in this report confirms that a pivotal step in
the proposed pathway of RBC senescence, ie, the denaturation and
deposition of globin on the membrane, occurs at the very end of the
RBC's life span. This result is in agreement with the data of Morrison
et al,4 who used a mouse hypertransfusion method to show
that RBC membranes from mice bind increased globin only at the very end
of their life span. Interestingly, the immunoblot of Fig 2 showed that
a majority of the globin which became associated with the membrane at
the end of the RBC life span was covalently cross-linked and resistant
to reduction with 100 mmol/L dithiothreitol. Therefore, the
cross-linked globin species may not be stabilized by disulfide bonds,
but rather by nonreducible linkages such as amides or free
radical-generated adducts, eg, bityrosine. Similar types of
nonreducible cross-linked globin species have been reported upon
treatment of Hb in vitro with free radicals.60-62
Because the oxidative denaturation of Hb might be expected to arise in
cells suffering from diminished intracellular reducing capacity, we
also analyzed the intracellular GSH concentration in several age
fractions of circulating dog RBCs. The observed late-term decrease in
GSH content of 70% appears to contradict previous studies examining
glutathione concentrations in senescent rabbit RBCs10 and
dense human RBCs.63 Besides species variation, one possible
explanation for the difference between our data and the data obtained
from the rabbit is that the dog RBCs deficient in GSH were collected at
a significantly older stage of the RBC life span than were the rabbit
RBCs. Specifically, the rabbit RBCs were assayed at 85% of their
total life span (60 days/total 70 days),18,19 whereas
dog RBCs were essentially at the end of their total life span (115
days). However, Piccinini et al63 isolated the most dense
0.4% of human erythrocytes and found no significant decrease in
intracellular reduced GSH. This difference between our data and their
density separated human RBCs may reflect the different RBC populations
sampled by density and biotin-dependent isolation techniques, or it may
alternatively reflect variations in the antioxidant systems used by
dogs and humans. More specifically, dog RBCs contain approximately
1/40 the catalase activity present in human
RBCs.64 This decreased catalase activity may cause an
increased reliance on glutathione peroxidase in the dog. Whatever the
explanation for this discrepancy between GSH levels in senescent human,
rabbit, and canine RBCs, the data appear to indicate that canine RBCs
suffer from severe oxidant stress late in their life span. The highly
cross-linked nature of the hemichrome-protein aggregates from dense
human cells33 would also seem to suggest that oxidative
stress is prominent in senescent human cells. Assuming GSH is not
diminished by such stress, it will be of interest to learn what
antioxidant system is compromised late in the human RBC's life span.
Based on our fluorescence-imaged microdeformation assay, it was
concluded that band 3 is less restricted by membrane-skeleton in
senescent RBCs than in younger RBCs. One possibility for this change is
that the detached population consists of otherwise normal band 3 molecules that have simply lost their ability to bind ankyrin as a
consequence of oxidation of cysteine 201 or cysteine 317.65 An alternative explanation is that the newly disjoined band 3 polypeptides comprise the fraction of anion transporters that have
already been clustered into microscopic aggregates by hemichromes. Because hemichromes compete aggressively for part of the ankyrin binding site on band 3,25,66 this aggregated population
would also be predicted to be free from skeletal constraints.
In the present study, we found that autologous IgG binding increased
abruptly at the very end of the dog RBC's life span. Dog RBCs greater
than 107 days old bound 150 molecules of IgG, whereas random aged
RBCs bound only 20 molecules. This sevenfold increase in IgG binding
agrees well with our preliminary study where RBCs only 104 days old
were examined.41 The data are also consistent with the
elevated IgG binding seen in human cells isolated by density
centrifugation2,33 and in mouse cells collected after
serial hypertransfusions.9 However, using the biotinylation
system to retrieve rabbit RBCs, Dale and
Daniels14 found no increase in the amount of
autologous IgG binding during the rabbit RBC life span. As we
have discussed earlier,41 the difference in IgG binding
between late-term rabbit and dog RBC's is most likely due to
species variations in the mechanisms governing RBC removal.
Although we did not attempt to examine the phagocytosis of senescent
dog RBCs, Singer et al9 have shown that RBCs isolated from
hypertransfused mice at the end of their life span are phagocytosed four times more aggressively than random-aged mouse RBCs. In addition, the densest fraction of human RBCs, which have been reported to contain
anywhere from 100 to 600 molecules of IgG per cell, have been shown to
undergo measurably increased phagocytosis in vitro.2 Depending on conditions used, it is generally accepted that anywhere from 100 to greater than 4,000 molecules of IgG must bind an RBC to
initiate phagocytosis.67-70 Furthermore, IgG subclass, the
cell-surface distribution of the antibody (clustered v
dispersed), and subsequent degree of opsonization by complement can
modulate the phagocytic response.39,52,67,71 Therefore,
although we have not performed a phagocytosis assay using our senescent
dog RBCs, we predict that the 150 molecules of IgG bound per senescent
dog RBC is sufficient to initiate their removal from circulation.
Determination of the exact progression of events responsible for
senescent RBC recognition and removal in vivo remains a difficult problem. However, the increase in membrane bound globin chains first
observed in cells isolated 86 days after biotinylation (Fig 2) occurs
at approximately the same time as the increased IgG binding and
decreased attachment of band 3 to the membrane skeleton. These
observations strengthen our hypothesis that induction of IgG binding to
senescent RBCs is tightly linked to the deposition of globin on the
inner membrane surface. Interestingly, whereas increased membrane-bound
globin was detected in a cell population 86 days old (mean cell age,
100 days), we found that intracellular GSH concentrations did not
begin to decrease until cells were 113 days old. This delayed
decrease in cytosolic GSH levels may suggest that its decline is more a
consequence than cause of globin deposition on the membrane. Indeed,
the oxidative stress accompanying Hb denaturation (for review, see
Hebbel and Eaton,72 and Chiu and Lubin73) could
potentially amplify a localized redox deficiency into a more global
cellular problem.
Although the deamidation of protein 4.1b to 4.1a has no known
functional consequences, it often is used to verify that a population of isolated RBCs is reaching senescence.5,74 Meuller et
al,5 using a serial hypertransfusion method to isolate age
defined mouse RBCs, were the first to show that protein 4.1b is
gradually converted to 4.1a over the course of an RBC's life span. Our
analysis of age-defined RBC membranes by gel electrophoresis shows a
nearly complete conversion of 4.1b to 4.1a in cells isolated 58 days after in vivo biotinylation. Because virtually all erythrocytes were
biotinylated on day 0, and because a partial cohort of young cells was
induced by prebiotinylation phlebotomy, a moderate fraction of the
biotinylated cells must have been no older than 58 to 62 days (mean
cell age, <87 days). Thus, the absence of any residual protein 4.1b
in cells isolated on day 58 was not likely a consequence of an
inexplicably old sample of biotinylated cells. Curiously, the half-life
for deamidation of asparagine 502 in the human has been reported to be
41 days.56 Since we see complete conversion of 4.1b to
4.1a during this same time frame, we would suggest that the deamidation
rate of asparagine 502 is accelerated in the dog and that dog RBC
populations enriched in protein 4.1a are not necessarily composed of
old RBCs.
Assuming asparagine 502 deamidation rates indeed differ among mammalian
species, what mechanism can be offered to explain this variability? Due
to the formation of succinimide ring intermediates,75 it
has been shown that asparagine deamidation rates are influenced by the
amino acid on the carboxy side of the asparagine
residue.75,76 Specifically, the rate of deamidation is
greater at asparagine-serine sequences than at asparagine-alanine
sequences.76 Inaba et al74 have
shown that deamidation of human protein 4.1 is consistent with these
criteria in that slow deamidation occurs at asparagine 502-alanine
during the human RBC life span. However, a serine immediately follows
asparagine 502 in the sequence of dog protein 4.1.55 Thus,
according to the rules governing asparagine deamidation, the rate of
conversion of dog protein 4.1b to 4.1a would be expected to be faster
than the conversion seen in humans.
In conclusion, the dog constitutes a useful animal model of human RBC
senescence because of its similar RBC lifespan and strongly age-dependent pathway of RBC removal. Although not all aspects of the
band 3 clustering hypothesis of RBC removal could be evaluated in this
model, many essential elements of the hypothesis were confirmed for the
first time in cells allowed to senesce in vivo to a known age. We
conclude that the opsonization of senescent RBCs, presumably at
hemichrome-stabilized clusters of band 3,24,31-34 still
constitutes a plausible pathway to explain the recognition and removal
of senescent RBCs.
| |
FOOTNOTES |
Submitted May 11, 1998;
accepted September 4, 1998.
Supported in part by National Institutes of Health Grants No. GM24417
and DK 26263.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Philip S. Low, PhD, Department of
Chemistry, 1393 Brown Bldg, West Lafayette, IN 47907.
| |
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Abstract
Introduction
