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A Second Generation Transgenic Mouse Model Expressing Both
Hemoglobin S (HbS) and HbS-Antilles Results in Increased
Phenotypic Severity
By M.E. Fabry, A. Sengupta, S.M. Suzuka, F. Costantini, E.M. Rubin, J. Hofrichter, G. Christoph, E. Manci,
D. Culberson. S.M. Factor, and R.L. Nagel
We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high
levels of human (I and PS chains ((1~/3'[p'"~I)
with a line
expressing human a and fiSAdmlh' (B'").
We hypothesized
that mice expressing both hemoglobins (Hbs) would have a
more severe phenotype becausethe reduced oxygenaffinity
and solubility of the
might enhance the rate and
extent of polymer formation. We obtained mice that expressed both @ and /3kAnti'lr.
The doubly transgenic mice
that are heterozygous for deletion of mouse
(Brn) occurred with reduced frequency andthose that are homozygous for deletion of mouse /3"jD' (BMDD)
occurred at a much
reduced frequency and suffered early mortality. Human (I
was 58% of all (I globin for all animals, whereas
and
pS"'"
were 34% and 28% of all j3 globins for
mice
and 4
2% and 36% for pMWmice. Hematocrit, Hb, and mean
corpuscular Hbwere normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice
versus ( 1 ~ / 3 ' [ / 3 ~ ~mice
1 older than 30 days (10.0% f 1.0% v
4.3% r 0.4%; P < .001, mean -c SE, n = 20 and n = 10,
respectively) andcontrol mice (3.9% k 0.4%). Reticulocytosis
was more severe in mice less than 30 days old (>20% for
(~~g'p~~@
mice).
" ] The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher
than that of ( 1 ~ / 3 " ~ ~mice
" ~ 1with a variable number of very
densecells.Delay times for polymerization of Hb in red
blood cells from ( 1 ~ / 3 ~ / 3 ~ ~ [ f f ' " Imice were shorter than
those of ( 1 " / 3 ~ [ / 3 ~mice,
~ ~ 1 and there were fewer cells with
delay times greater than 100 seconds. Urine-concentrating
a b i l i in control mice under ambient conditions is 2,846 f
294 mOsm and was reduced 30% t o 1,958 k 240 mOsm, P
c4 x
in all miceexpressing both transgenes.We conclude that doubly transgenic mice have a
more severe phenotype than either of the two parental lines. These mice may
be suitable for validating therapeutic intervention in sickle
cell disease.
Q 7 9 9 5 by The American Society of Hematology.
A
mouse pMaJo'
(Hbb'"" '), which we symbolize as p"" or p"""
to increase expression of the p globin transgenes.
The SAD mouse4.' incorporates all three mutations (the
sickle mutation plus the Antilles and D-Punjab) in one p
chain and is the product of site-directed mutagenesis grafted
onto a S-Antilles /3 gene. Mice that are also heterozygous
for the mouse pMajor
deletion express this trimutated chain
at moderate levels (29% p"") and are characterized by anemia in the neonatal period, but notin the adult mice. In
addition, they die after short exposure to hypoxia' and suffer
from high intrauterine mortality.
The efforts reported here had two goals: (1) to develop a
transgenic mouse model that is intermediate between the
mild-to-moderate phenotypes and the severe SAD mouse
and (2) to test the hypothesis that the pathology observed
/3"'"
/
3
'
N ANIMAL MODEL for sickle cell disease has been
a long sought goal that has recently been realized in
several laboratories by the generation of transgenic mouse
lines expressing hemoglobin S (HbS) or derivative forms of
this mutant Hb."6 The animals generated have a range of
pathophysiology from severe phenotypes that resulted in increased perinatal mortality to lines that exhibited moderate
or mild pathology under ambient conditions.
Two naturally occurring mutations increase the severity
of HbS-related disease. One of them is HbS-Antilles, which
contains, in addition to the HbS mutation at p6 (Glu-rVal),
a second mutation in the same chain at p23 (Val+Ile). HbSAntilles has low oxygen affinity and low solubility under
deoxy conditions. In contrast with sickle trait individuals, the
heterozygous individuals with HbS-Antilles have significant
pathology. The other mutation is HbD-Punjab, which occurs
at p123 in a gene that does not contain the sickle mutation.
This trans mutation results in pathology when the patient is
a double heterozygote for both PS and ,L?D-PunJab because of
assembly of tetramers that contain one p chain from each
of the two mutated genes.
Both of these mutations have beenused
to create
transgenic mice. Transgenic mice using the S-Antilles mutation were generated by Rubin et a13 and were found to have
a slightly reduced hematocrit and a small number of irreversibly sickled cells (ISCs) that increased when the animals
were subjected to hypoxia (a hypobaric chamber at 0.42 atm,
which is equivalent to 8.4% 02).
The animals studied, which
were homozygous for mouse PMaJor
deletion, expressed about
5 1% of their &globin as flS-Antil'es;
however, only 10% of the
a chains were human a (aH),
and therefore, most of the
flS-Antilles
was found in dimers that contained mouse a-globin.
As shown by Rhoda et al,' mouse a chains interfere with
polymerization nearly as effectively as do human y chains.
All of the mice described in this report make use of animals
either heterozygous or homozygous for the deletion of the
Blood, Vol 86, No 6 (September 15). 1995 pp 2419-2428
From theDepartments of Medicine and Department of Pathology,
Albert Einstein College of Medicine, Bronx, NY; the Department of
Genetics and Development, Columbia University, New York, NY;
the Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, Berkeley, CA: Laboratory of Chemical Physics, NlH,
Bethesda, MD: and the Centralized Pathology Unit for Sickle Cell
Disease, University of South Alabama, Mobile, AL.
Submitted November 2, 1994; accepted April 13, 1995.
Supported in part by National Institutes of Health Sickle Cell
Center Grant No. HL-38655 and a Grant-in-Aidfrom the New York
Branch of the American Heart Association.
Address reprint requests to M.E. Fabry, PhD, Albert Einstein
College of Medicine, Department of Medicine, Vllmann 921, 1300
Morris Park Ave, Bronx, NY 10461.
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.
Q 1995 by The American Society of Hematology.
0006-4971/95/8606-0143$3.00/0
2419
2420
FABRY ET AL
under hypoxic conditions in the aHpS[pMDD]
mouse is caused
by polymerization of HbS.
To achieve this end we have bred the transgenic mouse
line aHPS[PMDD],
which has a phenotype with moderate pathology under ambient conditions and more severe pathology
under hypoxic condition^,'.^ to the aH/3S-Anti11es
line. The rationale is that the combination of the low oxygen affinity, low
solubility HbS-Antilles, and the high level of human (Y and
PS expression of the aHpS[pMDD]
line might result in an
enhanced rate and extent of polymerization and a more severe phenotype than either of their parents.
MATERIALS AND METHODS
Hematologic Parameters
Mice were bled from the tail using protocols approved by the
animal studies committee and blood samples were analyzed for reticulocytes using the Sysmex R-1000 system (Sysmex, Toa, Japan) or
by staining with thiazole orange and evaluating by fluorescenceactivated cell sorting (FACS) (Lysys 11; Becton Dickinson, Mountain
View, CA). Serum samples were analyzed using the Chem-l system
(Technicon, Tarrytown, NY).
High-Performance Liquid Chromatography (HPLC)
The globin composition was determined by HPLC using a denaturing solvfnt that separates the globin chains and a Vydac largepore (300 A) C, column (Separations Group, Hesperia, CA), 4.6 X
250 mm with a modified acetonitrilekl,O/trifluoroacetic acid gradient similar to that used by Schroeder et all0 for separating human
chains. Two buffers were used, A (0.18% trifluoroacetic acid in 36%
acetonitrile) and B (0.18% TFA in 46% acetonitrile). Starting with
38% B, the percentage of B was increased by 0.583% per minute
until all of the globin chains were eluted.
Slow Deoxygenation
Blood samples were collected directly from the mouse tail into
heparinized capillary tubes. The samples were carefully mixed in
the tubes and then immediately sealed on both ends with tubesealing compound. They were allowed to sit at room temperature
for approximately 24 hours. The hematocrit tubes were then placed
in a nitrogen-filled glove bag where the ends of the tubes were cut
off. By using a piece of small bore plastic tubing attached to the
end of a needle and syringe, the cells were pushed out into vials
containing degassed 2%glutaraldehyde in phosphate-buffered saline,
pH 7.4.
CSAT
Blood from several mice with identical HPLC chromatograms
was collected and washed into saline. The cells were hemolyzed by
the addition of distilled water and by rapidly freezing and thawing
several times. They were spun down in a microfuge and the clear
red supernatant removed. The resulting hemoglobin was dialyzed
and concentrated versus 0.1 m o m potassium phosphate buffer, pH
7.35 at 25°C. Samples were deoxygenated on ice by adding enough
sodium dithionite solution to give a final concentration equal to
three times the heme concentration. The samples were transferred
anaerobically to CsATtubes filled with paraffin oil. They were incubated at 25°C overnight in a nitrogen atmosphere. The mouse Hb
samples were centrifuged for 2 hours at 35,000 rpm at 25°C with
purifiedHbSused
as a control. The supernatants were removed
anaerobically and concentrations and deoxy pHs were determined.
Density Gradient
The density gradient screening ofredblood cells (RBCs) was
performed by Percoll/Larex gradients." Percoll (colloidal silica
coated with polyvinylpyrrolidone; Pharmacia, Upsalla, Sweden) and
Larex (arabino galactan polysaccharide; Larex International, Duluth,
MN) gradients were formed from a mixture of Percoll and Larex
(density 1.207 g/mL, determined from refractive index), water, and
10-fold balanced salts in a ratio of3.5:3.0:2.8:0.7. The pH and
osmolarity are adjusted to 7.35 and 330 mOsm, respectively. Batches
are prepared and frozen in 50-mL aliquots. A 0.1-mL aliquot of a
well-mixed sample of whole blood with the hematocrit adjusted to
50 was added to 5.9 mL of the gradient mix for analytical determinations of RBC density distribution. The tubes are well mixed, and
spun in a rotor (model 5-21; Beckman Instruments, Irvine, CA) for
25 minutes at 35,000g at 37°C. The resulting gradient is continuous
but nonlinear. Each group of eight tubes included a sample from a
control mouse and a tube with Pharmacia density-marker beads to
ensure constant conditions. Scrupulous care in resuspending blood
samples at all stages is critical in these measurements because the
densest cells selectively adhere to the bottom of the tube.
Delay Time
Delay times were measured for three aHps/?s-A"t[pMD]
mice and
mouse under conditions that are physiologic
one aHpsps~A"t[OMDD]
for mice (pH 7.35 and330 mOsm). The plasma osmolarity characteristic for these mice was determined by 16 separate measurements
on control C57BLJ6J mice in which we found an average plasma
osmolarity of 331 2 16 mOsm. A similar value was found in the
literature."
Mice were bled approximately 1 hour before the delay time measurement was made; the cells were washed into a physiologic buffer
(330 mOsm for mice") and diluted to Hct 0.1. The cells were then
equilibrated with a humidified mixture of CO and COz at 37°C until
pH 7.35 was reached (by adjusting the percent CO,) and transferred
into a closed Dvorak-Stotter cell. The RBCs were allowed to settle
in the cell while maintaining a temperature of 37°C for 20 minutes
until most of the cells were adherent to the polylysine-coated glass
surface. After this, the cell was inverted over a microscope. and the
CO was flashed off the Hb by exposure to a focused laser beam at
514.5 nm. Onset of polymer formation was measured by continuously recording light scattering as previously described.l3.I4Although
the actual measurements were more difficult because of the small
size of the mouse RBCs, the polymerization curves obtained from
mouse RBCs by this protocol were similar in shape and magnitude
to those obtained from human RBCs.
Urine-Concentrating Ability
Because of the greater fragility of pS/lps-""'
mice, they were deand four
prived of water for 7 daytime hours. Six aHps/?S-A"t[pMD]
a"~s~s~A"'[/?MDD]
mice were examined. This less rigorous protocol
(usually the mice are deprived of water overnight) allowed us to
observe the animals and rescue any animals showing signs of lethargy or listlessness. At the end of this period urine was collected
onto Parafilm and the osmolarity was measured after a 1:lO dilution
with distilled water.
Pathology
A total of 7 aHpspS-""'[lpMDD]
mice were examined; 4 of the animals (ages 87, 88, 151, and 157 days) were killed during the course
of experimentation, 2 died spontaneously, and 1 was killed because
it appeared tobe terminal (30, 91, and 103 days). Tissues were
preserved in buffered formalin. Slides were stained with hematoxy-
2421
A TRANSGENIC MOUSE WITH HbSPLUSHbS-ANTILLES
Table 1. Hematologic Properties of Transgenic Mice
Reticulocytes %
(age > 30 dl
Mouse
C57BU6J
(n = 6 )
U
MCV
MCH
MCHC
IfL)
(pg/cell)
lg/dL)
3.9 f 0.4
45.4 f 0.9
14.5 2 1.0 33.7 ? 0.4
4.3 f 0.4
43.0 ? 1.4
14.1 2 0.7
35.7 2 1.7
14.2
34.6 2 1.5
W
LLY
a H p S l p D 1
(n = 10)
(IHpS.Ant[ pMDD]
(n = 5)
,HpSpS.Am[
5.1 t- 0.7
43.6
f 0.3
I
0.9
pMD]
(n = 9.5
4)
f 1.2
42.2 ? 1.8 14.4 2 0.2
36.2
2 1.1
,HpSpS-Am[pMDD]
(n = 22)
10.0 2 1.0
43.5
f 1.5
14.9 I
0.5
36.2 2 0.7
Abbreviation: MCV, mean corpuscular volume.
lin-eosin or trichrome. The reported results arefor the animals killed
during the course of experimentation.
RESULTS
Hematology and Hb Composition
Hematocrits, Hb,
and
mean
corpuscular hemoglobin
(MCH) were normal for all adult mice. Mice bearing both
transgenes expressed both 8' and p'"'nlilles
as well as human
a;however, the percent of PS and fl'-Anlillcr expressed as the
percent of all p chains for both DMDand OMDDmice was less
than that expressed by either parent that was homozygous
for the pMaJordeletion
(pMDD)
(Table 1). Expression of human
a was 58% ? 1% of all a globin for all animals, whereas
the expression of p' and pS"'"i''es
was 34% 2 2% and 28%
5 1% of all p globins for PM"mice and 42% f 3% and
36% ? 2% for DOMDD
mice (Table 2).
aHpSpS-Ant
[PMDD]
Fig 1. Percoll-Larexcontinuousdensity
gradient with density
mouse, an
marker beads, a control C57BLK.J mouse, an uHpS[pMDDl
a"pspsAmIpMDI
mouse, and an
( ~ ~ p ~ p mouse.
~ - ~Notethe
~ [ p retic~ ~
ulocytes (low-density cells at the top of the tube) in both of the
the high-density cells in the #f3*p"
doublytransgenicmiceand
[pMDD]
mouse.
~ ]
Sickling
Cells from aHflSfl"A"'[PMDD]
mice were allowed to sickle
slowly
and
were
subjected
to
scanning
electron microscopy
Density gradients (Fig 1) were completed for control
(Fig 2). More than 95% of the cells sickled and many exhib(C57BW6J). aHfl'[fiMDD],
aHPSPS-Ant[pMD],
and c~~fl~fl'-~"'
ited a profusion of spicules.
[pMDD]
mice. As previously reported, the aHfl'[pMDD]
mice
had a mean corpuscular hemoglobin concentration (MCHC)
CS*7
that is about 3 g/dL higher than that of the control mice.
Both ,Hpqp5-Anl[pMD] and a H p S p S A n l [pMDD]
mice also had
The concentration of the Hb in equilibrium with the deoxy
denser cells than control mice; in addition, they hada populagel phase was determined for aHfl'[pMDD],
aHpsps-Anl[fiMD].
and aHpSpS-Anl [pMDD]
tion of low-density cells that were predominantly reticulotransgenic miceandwascompared
cytes, and aHflSfls"'nl[pMDD]
mice had a population of higher
with mixtures of HbS with HbAor HbF (Table 3). We found
density cells. The aHflSfl'"'"l[PMDD]
mice had the largest perthat the CSATfor aHpps-A"l[flMDD]
mice was nearly 3 g/dL
cent of high-density cells seen in any of the mice that we
less than that of the aHpS[[BMDD]
mice. Althoughthese values
have examined to date.
are only slightly less than those for a sickle trait patient, the
tendency to form polymer in vivo will be higher because of
the lower p50 of S-Antilles and the higher MCHC of mouse
RBCs.
Table 2. Hemoglobin Expression of Transgenic Mouse Lines
Density Gradient
Mouse
0' + /3SAm/All0
Os/All 0
2 2.4
74.7 f 2.4
74.7 2 2.4
-
11.1 2 2.7
50.9 2 2.3
-
50.9 2 2.3
2 1.4
62.6 I
2.1
34.4 f 2.0
28.3 2 .8
? 2.3
42.2 I
2.8
35.9 2 1.7
aHIAll a
Os.A"'lAll0
Delay Times
Uy?S[
(n = 20)
55.9
,HpS.Am[pMDD]
(n = 4)
,HpSpS-Am[
pMD]
(n = 8)
57.8
,HpSpS.Am[
In = 10)
pMDD]
58.2 2 78.2
.9
The time between deoxygenation and the onset of polymer
formation was measured using laser photolysis of CO-Hb to
produce deoxy-Hb followed by detection of polymer formation by light scattering. Three mice with aHps~"-"'[pMD]
and
one mouse with aHflsp'-Ant[pM"D]
were tested and compared
with three mice with the aHp'[flMD"]
genotype (Fig 3). We
found that the major difference was in the number of cells
FABRY ET AL
2422
Fig 2. Scanningelectronmicrograph RBCs
of
from a
,HpSpS.Ant[pMDD]
mouse that
have been deoxygenated for 24
hours. Note the multispiculated
forms, which are not common in
aHpS[pMDDi
mice.
requiring 100 seconds or more to formpolymer. In mice
with the a"~Sps"'n'[~MD"]
phenotype, more than 95% of the
cellssickled in lessthan 50 seconds,whereas in al'flSflS"'"'
[pM"]and aHps[[pMD"1
mice, 16.5% and 39%. respectively,
requiredmorethan 100 seconds to sickle. Weknowfrom
previous experiments that more than 95% of the cells will
sickle if maintained under deoxy conditions for long enough,
thus even though these cells required more than 300 seconds
to form polymer, they are able to sickle. Less obvious in the
cumulative plots used are the cells withveryshort
delay
times (between 1% and 5% at .01, .OS, and . l seconds) of
the aHp?p?-l\rU
[pMD]
and the aHp"pS"'"'[pM""l
mice.
hand-counting methods, but that the results were much more
reproducible, in part because of the much larger number of
cells used for each determination. Adult ~ r " p ' p ~ " ' " ' [ ~ ~ " ]
micehad increased levels of reticulocytes (Table 1 ) and
adult ~ r " p ~ ~ ~ " ' " 'had
[ p "levels
" ~ ] of reticulocytes higher than
either parentline. Reticulocyte levels werehigherforthe
doubly transgenic mice versus aHp'[pM""]
mice older than
30 days (10.0% t 1.0% 11 4.3% t 0.4%, P < .001, mean t
SE, n = 20 and n = 10. respectively) and control mice (3.9%
t 0.4%). This effect was even more marked in mice under
30 days of age in which the ~ r ~ ~ ~ ~ ~ "mice
' " were
' [ ~ ~ " " ]
found to have 20% reticulocytes (Fig 4).
Reticulocytes
Urine-Concentrating Ability
Reticulocytes were measured usingthe automated Sysmex
system and thiazole orange with FACS analysis. We found
that there was a consistently lower percentage of reticulocytes recorded with theSysmex system when compared with
Urine-concentrating ability after a 7-hour-long deprivation
of water was reduced under ambient conditions in the PS/
fls"'"' mice that were either hetero- or homozygous for the
mouse P""J'"deletion when compared with control mice (Fig
5). The values obtained were more varied because
of the
shorter period of water deprivation, butthemore
fragile
nature of the pslps"'"'
mice dictated this protocol. Reduced
urine-concentrating ability is a characteristic of sickle cell
disease and urine osmolarity was reduced 30% to 1,958 Z
240 mOsm under ambient conditions in all mice expressing
both transgenes ( P < 4 X IO-'; 2,846 t 294 mOsm in
control mice).
Table 3. CsAT for SamplesContaining p"
Sample
C s ~ gldL
r
Purified HbS
HbS/HbA 40:60
HbSlmouse 50:50
HbS/HbF 50:50
17 2 1
27 2 2
31 2 1
30 2 1
28.9 t 1
34+
27.2 2 1
25.9 2 1
a n p 1p D D I
aHflS-Ant[
p D D ]
aHljSpS.Anl
nHpflS.Am[
[P
D I
p D D ]
4(mix - HbS/HbA 4060) g/dL
-10
0
4
3
1.5
J+
0
-1.1
Serum Enzymes
Two serum enzymes associated with liver damage were
analyzed: aspartate amino transferase and alanine amino
2423
A TRANSGENIC MOUSE WITH HbS PLUS HbS-ANTILLES
transferase. For aspartate amino transferase, the aHPSPS-Ant
[pMDD]
mice had an average value of 167 -C 23 U/L versus
the control C57BV6J mice, which had 52 2 4 U L (mean rC_
SE, P < .002, n = 15 and n = 8, respectively). For alanine
amino transferase, the aHpSpS-An'[/3MDD3
mice had an average
value of 166 32 U/L versus the control C57BV6J mice,
which had 27 2 1 U/L (mean 2 SE, P < .005, n = 15 and n
= 8, respectively). Mice heterozygous for the pmajor
deletion
aHPSPS-Ant[pMD]
had intermediate values (Fig 6).
I
25t:
0
Pathology
A total of seven aHps~s~A"'[pMDD]
mice were examined
using hematoxylin-eosin and trichrome stains. The results
reported are, unless otherwise indicated, for the four animals
that were killed during experimentation, but the other three
animals had very similar and, in some cases, more severe
pathology.
Brain. The two younger animals had congestion, occasional red neurons and rare pyknotic neurons. Red neurons
were also seen in three other aHpSp"-""'[pMDD]
mice. The red
neurons may be indicative of hypoxic episodes secondary to
focal vasoocclusion; however, they would not be regarded
as significant in the absence of the more severe findings in
the two older animals. The two older animals had neuronal
dropout, pyknotic neurons and supporting cells, and pyknotic
Purkinje cells in the cerebellum (Fig 7A).
Lung. The lungs of the two older animals manifested
0
10
20
30
40
c.
c
50
a"!
60
70
80
90
100
l
l
.01 .05
l
l
.l .5
l
l
'
5
l
l
10
l
t
50 100
-
L
c300
Delay time in seconds
Fig 3. Cumulative delay times for three &""~"DD1 mice, three
a"/3s/3sA~"Dl
mice, and an aHf3'/3sAR[BMwI
mouse. Note that the
aH/3'/3"~/3""l mouse (0)has more than 95% of cells with delay
times (the time between complete deoxygenation and the onset of
polymer formation) less than 50 seconds. In theC Y " ~ ' / ~ ~ ~ " mice
~~"I
(01 and &"'" [p-]
mice (A). 16.5% and 39%. respectively, of the
cells required more than 100 seconds to sickle. In addition, note that
both the aH/3'~"""1/3"l and the a"/P/3sM1~01
mice have more
cells with short (less than 0.1 second) delay times, although this is
hard to see in cumulative plots.
01
0
I
I
l
I
I
50
100
150
200
250
I
I
I
300 350 400 450
Mouse age in days
Fig 4. Percent reticulocytes versus mouse age
in days: aH/3s@MDD1
(0)
and a"/3s/3BM"vMDDI
(0).Note thevery high reticulocyte count of
a"p'ps~A"[f3MDD1
mice at 30 days of age.
septal thickening (Fig 7B) and interstitial fibrosis with some
platelet thrombi. Severe congestion and intra-alveolar hemorrhage were noted focally in all four animals.
Liver. In all cases examined, we found multifocal ischemic infarcts of ages varying from recent (1 to 2 days old)
to remote (several days to several weeks old) with dilatation
of central veins and a preserved rim of viable cells around
the central vein, suggesting recent andor prolonged ischemic
events (Fig 7C). One of the older mice showed evidence of
past infarcts, but did not have evidence of recent infarcts.
Areas of recent infarct featured focal coagulative necrosis
with degenerating hepatocytes showing nuclear chromalysis
and smudged cytoplasmic details with ghost-like outlines of
hepatic plates and sinusoids. The areas of remote ischemic
injury featured complete coagulative necrosis with loss of
nuclei and amorphous eosinophilic outlines of hepatocytes
and sinusoids. This is a new feature that has not been reported in other sickle transgenic mice. Occasional Kupffer
cells could be visualized in the trichrome stained slides with
RBC fragments (not shown). There was no appreciable iron
deposition in the liver, but scarring and fibrosis was seen in
older animals. Figure 7C illustrates old and new lesions in
the liver. In the two older animals, fibroticand collapsed
areas were seen. The liver was enlarged in aHPSPS-Ant[PMDD]
mice versus C57BW6J of the same age (6.3% t 0.7% of
body weight v 4.8% t 0.8%, n = 8 and n = 5, respectively;
P < ,009; Table 4).
Spleen. Spleen to body weight was calculated for eight
animals and was enlarged in aH/3sps-Ant[pMDD]
mice versus
C57BL/6J of the same age (0.68% 5 0.27% of body weight
v 0.26% f 0.04%,n = 8 and n = 5 respectively; P < .W6
Table 4) with significant fibrosis (visualized with trichrome
stain, not shown), marked congestion with an expansion of
2424
FABRY ET AL
red pulp, but without excessive iron accumulation. Focal
350
fibrosis was noted in the two older animals (15 1 and 157
days).
Kidney. The kidneyhad congested glomeruli andthe
afferent vessels were dilated. There was focal fibrosis in the
medulla and the cortical-medullary junction, but only one
6 250
U)
animal had fibrosis in the cortex. Glomerular and peritubular
e
S!
vessels were markedly congested as were vessels in the paU)
200
pilla. The kidney was enlarged in aH,@@S-Ant[pMDD]
mice
l
versus C57BL/6J of the same age (0.85% -C 0.12% of body
weight v 0.57% ? 0.03%, n = 8 and n = 5 respectively; P
'z
150
U
< .0005; Table 4).
0
AspAmTx
0
5
e
8
+-.
Percent
ps/ps~*"'
Mice
and Length of Survival
;
100
a
Fewer aH~SPS-Ant[PMDD]
mice survived to 10 days than
were predicted. Mating aH/3s[[pM"D]
micewith
aHPS-Anl
50
[pMD"]
mice wouldbe expected to produce 25%of four
different genotypes: aHpS[pMDD],
aH/3S-An'[pMDD],
(-)[pMD"]
01
l
I
I
I
(homozygous for pmaJo'
deletion, but no transgene), and
aHPSPAnt
[p"""], but 28.6%, 34.9%, 27%, and 9.5% were
found instead (P < .03, n = 63). Not all aHpsps-An'[pM"D]
mice were produced from this type of mating. In contrast,
Fig 6. Aspartate amino transferase ( 0 )and alanine amino transmating two aHpS[pMDD]
mice produces 73% aHps[[p"""] ferase (0)
in U/L for control (C57BI).n"ps [pmD],
a"pSB6"*tpMDl,
and
c ~ " p ~ p " ~ "mice.
l
All animals were maintained under ambient
mice and
27%
(-)[pMDD]
mice; indicating that
the
conditions. Ages range from48 to 271 days.
aHpS[pMDD]
transgenic phenotype is more robust than the
homozygous /?"'J"'deletion. Of 22 aH/3sps-Ant[pMDD]
mice
3200
3000
2800
2600
2400
2200
2000
1800
1600
Fig 5. Urine-concentrating ability for control mice (males, e; females, 0)and p/p"* mice after a 7-hour deprivation of water.
Female mice ara slightly
offset to the right. Theps/psh mouse group
contains animals
that are both homozygous(0.all males) and heterozygous (males, females, 0)for the mouse p""'" deletion.
+;
surviving to 10 days, 5 died of natural causes between days
20 and 138, 1 additional mouse died accidentally during the
course of experimentation, 8 were killed during the course
of experimentation, and the 2 oldest surviving mice are 1
year old. Thus, more than 20% of these mice died before 5
months of age, which is in contrast with the 30 months that
is normal for aHpS[pMDD]
mice and C57BU6J mice.
DISCUSSION
We have previously reported on transgenic mice that express high levels of human a and PS (aHpS[pMDD]).5,6
We
found that these mice had pathology under ambient conditions that could be made more severe when the mice were
subjected to hypoxia'; therefore, we concluded that the enhanced HbS polymer formation induced by hypoxia resulted
in increased vasoocciusion. If this interpretation is correct,
mechanisms that increase polymer formation should result
in increased pathology. To test this hypothesis and create a
mouse with a more severe phenotype, we have introduced
/3S-Anti"es into
.
the aHpS[pMDD]
line, examined indicators of
polymer formation, and compared these with hematology,
urine-concentrating ability, and histopathology.
The rate of polymer formation is extremely sensitive to the
concentration of deoxy Hb, with a concentration dependence
exceeding the 30th power,'',16 whereas the extent of polymer
formation at equilibrium is directly proportional to the
deoxy-Hb concentration. If polymer formation occurs slowly
(with a long delay time), cells may be reoxygenated in the
lung before polymer formation occurs. Delay times that are
long with respect to the mean time of circulation would be
expected to protect the animal under most circumstances;
however, if adhesion or stasis occurs or the transit time is
NSGENIC
A
2425
Fig 7. Hematoxylin-eosin-stained sections of brain (A), lung (B),and liver (Cl. In (A), cerebellum from a 151-day-old ~~"&3""rp"l mouse
showing pyknotic (filled arrow) and normal (open arrow) Purkinje cells. The pyknotic cells are hyperchromaticwith shrunken nuclei and are
adjacent to cells with normal nuclei, consistentwith focal vasoocclusion. Each Purkinjecell is served by a single capillary (curvedarrow) and,
hence, focal loss of them cells is an expected result of sickle cell vasoocclusion of single capillaries. One such capillary that ispacked with
cells is indicated by the curved arrow (original magnification x 160). In (B),lung parenchyma with alveolar capillary congestion and focally
thickened alveolar septa(arrows)from a 157-day-oldaHpsfis.h[fiMDDl
mouse (original magnification x 160). In (C), a low-power view (original
magnification x 6 5 ) of the liier is seen on the right
showing multiple infarcts; the boxed areas areshown at higher magnification. The top of
(C) (a) shows a recent ischemic injury with partial nuclear chromalysis; the bottom of (C) (b) shows a more remote ischemic injury with
coagulative necrosis of hepatocytes(original magnification x 280).
low solubility of HbS-Antilles leads to a lower CsATand
very long, the delay time will be less important than the
shorter delay times for polymerformation in RBCs in
ability of the cell to form polymer. Intracellular polymer
these transgenic mice. All of these properties interact to
formation results in rigid, nondeformable cells with inproduce enhanced vasoocclusion, RBC destruction,
and
creased viscosity and potential
for vasoocclusion,'7 and
pathology, increased spontaneous death and, in the case
therefore, both intrinsic and extrinsic factors that affect
of the aH/3S@S-Ant[/3MDD]
mice, a birthrate that is half of
polymer formation are expected to affect pathology.
that expected.
We havegenerated and studiedtransgenicmicethat
The amount of Hb per RBC is constant for all mice regardexpress both PS and @S-Antilles
on a background which is
less of whether they are C57BV6J control mice or express
either
heterozygous
forthe
pMaJor
deletion ((.u"@~@~one or two transgenes. Consequently, although we do not
A"'[pm])or homozygous for thedeletion
([(.u"@~@~know if this down regulation occurs at the level of transcripht[@MDD]).
We find that these mice have a more severe
tion, translation, or polypeptide chain assembly, the expresphenotype than either of the two parent lines. Features of
sion of more than one transgene will result in reduced expresthese mice that will lead to an increased concentration of
sion of each of the individual transgenes.
deoxy PSand pS-Aatilles
are (1) higher percent of human a
A major difference between human sickle cell disease and
and the sum of the mutated @-globin chains (Ps plus p"
all transgenic mouse lines described to date is the absence
Anti'les); (2) the decreased oxygen affinity of @S-Ao'i11es,which
of adult anemia. Both human and animal studies indicate
results in the presenceof more deoxy @S-Antil1es;
and (3) the
that the low hematocrit characteristic of sickle cell disease
greater degree of dehydration of the RBCs of the doubly
is protective against vasoocclusive events, and that increased
transgenic mice, which increases the intracellular Hb conhematocrit results in greater pathology because of increased
centration. The combination of these properties with the
2426
FABRY ET AL
Table 4. Organ Weights for Control and Transgenic Mice
cytes were elevated compared with the parent lines. This
effect was particularly intense in mice younger than 30 days
of age. In the neonatal mouse, RBCs are larger than those
C57B1/6J
5
135
2.6 2 0.4
48.0 t- 1.6
5.7 2 0.3
of the adult and the level of 2,3-DPG expression is less than
aH@I p D D 1
9
147
5.7 t 3.1
55.6
2 1.7
7.4 t 1.1
aHpSpS-Ant
that of adult mice2’; in control mice, adult values for both
[pMDD] 8
131
6.8 2 63.3
2.7
2 8.5
6.6
2 1.2
of these properties are attained by the end of the first 4 weeks
Pcontrol v
pSpS-Ant
of life.” One possibility is thatone of these two effects (large
<.006
<.009
1.0005
size or reduced 2,3-DPG) is responsible for the enhanced
* Mouse age in days.
rate of RBC destruction. In the aHflSPS-Ant[pMDD]
mouse, the
t Percent of body weight x 10.
greater number of dense cells (Fig 2) may contribute to the
increased reticulocyte count.
Density gradients of human patients homozygous for HbS
bulk blood viscosity. Thus, a low hematocrit in a transgenic
(SS)
have a large and variable population of dense RBCs
animal model wouldresult in less sickling-related pathology.
that is composed of both dense discocytes and ISCs.” The
Nevertheless, the probable origin of the absence of anemia
mechanism by which this population is formed is still not
needstobe considered. In transgenic mice, a mechanism
fully resolved, but it is generally agreed that it is the result
contributing to the absence of anemia is the relative right
of repeated cyclesinof
vivo
which results in
shift of the oxygen equilibrium in mouse RBCs (because the
potassium loss and dehydration. Density gradients of
host Hb has a higher ~ 5 0 ~Another
).
major component of
aHps@s^nt[pMDD]
mice indicate the presence of both a large
the level of hemolysis in human sickle cell disease that is
reticulocyte population and a much larger population of high
absent in the mouse is the formation of very dense cells with
density RBCs than i s seen for either of the parent lines.
a life span of only 2 to 4 days. We have reported the presence
Romero et all8 have shown that deoxygenation of transgenic
of a deoxy potassium efflux, a unique characteristic of human
mouse RBCs results in a deoxy potassium effluxthat is
sickle cells, in RBCs of the aHPs[[PMD”]
mouse that would
analogous to that uniquely observed in human SS patients.
be expected to contribute to formation of dense cells. Howde
Francheschi et a124have recently reported a Ca2+-stimuever, the K:CI cotransport detectable inthe mouse lacks
lated K+ channel in the SAD mouse analogous to that obmany of the characteristics found in human RBCs, and the
served by Romero et a]’* in theaHpS[pMDD]
mouse that probhigh activity of calmodulin-activated Ca2’-ATPase (which
ably
is
responsible
for
the
deoxy
K+
efflux
in both cases.
is correlated with Ca2+-pumpactivity) in mouse RBCs may
An increased population of high-density cells is seen in
partially protect PS transgenic mouse RBCs from dehydraaHps[[pMDD]
mice that have been exposed to hypoxia5;in this
tion even inthe presence of a deoxy potassium efflux.” This
case, the increased population was attributed to increased in
may account for the small proportion of ISCs found in all
vivo sickling. Similarly, we speculate that the increased RBC
transgenic mouse models19and will also contribute to the
density seenin the aHpsps~Ant[pMDD]
mouse is caused by
absence of adult anemia.
nesum of pS and pS-Antilles In. aHpsps-Ant[/?MDD]
mice is
increased in vivo sickling under ambient conditions.
The time between complete deoxygenation and the onset
close to 80% compared with 72% PS in our previous line,
of
polymer formation (delay time) was measured in individaHpS[pMD”].
Because the human aHexpression is higher in
ual RBCs for aHpspS-“”‘[PM”]
and aHPSPS-Ant[pMDD]
mice
these animals (close to 60%) compared with the S-Antilles
and compared with the parent aHps[pMDD]
mouse Iine (Fig
mice and the sum of PS plus pS-Antilles
is higher than the
3). We found that both the aHPSPS-Ant~MD]
and the aH,BSPS-An‘
expression of PS in the aHpS[,BMDD]
mouse line, we antici[pMDD]
mice
had
more
cells
with
delay
times
less than 100
pated and found that the concentration of deoxygenated Hb
msec and fewer cells with delay times greater than 100 secin equilibrium with the polymer (CSAT) for the aH@SpS-Ant
onds than the aHpS[pM””]
mice; however, in aHfls@s-An‘
[pMDD]
mouse is less than that for either of the parents and
[P”””]
mice,
95%
of
the
cells
sickled in 50 seconds or less.
less than that for a mixture of HbS and HbA similar to that
It
is
not
known
whether
the
deleterious
effects of a small
found in sickle trait RBCs. The physiologic impact of this
percent of cells with very short delay times is moreimportant
CsAT must
be interpreted in the context of other RBC properthan the protective effect of a larger percent of cells with
ties; relevant here is the increased MCHC of transgenic
long
delay times. Although the aHpS,f?S-Ant[,BMD]
mice had a
mouse RBCs under physiologic conditions, resulting from
smaller
percent
of
polymerizable
Hb
plus pS-Antille.i)
dense cell formation and the high normal plasma osmolarity
than the aHps[[pMDD]
mice, they had shorter delay times that
in mice (330 mOsm). Therefore, the same CsAr will result
appear to be a contradiction. The explanation for this disin a shorter delay time in transgenic mouse RBCs than it
crepancy canbe found in the properties of HbS-Antilles.
will in human cells. Furthermore, CsAT and delay times are
HbS-Antilles differs from HbS in both its decreased oxygen
measured under fully deoxygenated conditions and will not
affinity (9 mm Hg v 5.5 mm Hg, respectively”) and in its
reflect the important in vivo effect of the reduced oxygen
decreased solubility in the deoxy state (1 1 g/dL versus 18.4
affinity of cells containing PS-Antilles.
g/dL, respectively”). In vivo, we expect oxygen affinity to
In adult mice, increased reticulocyte counts are an indicaplay a strong role, because RBCs are usually only partially
tion of increased erythropoiesis and when they are combined
deoxygenated, and the low p50 of HbS-Antilles will increase
with an analysis of spleen histopathology, reticulocytosis can
the concentration of deoxy Hb present and hence shorten
be attributed to an increased rate of RBC destruction. In
delay times. However, under the experimental conditions
adult aHpsps-An‘[pMD]
and aH@5PS-Ant[PMDD]
mice, reticuloMouse
Kidneyt
LivertSpleent
n
Age*
vs
A TRANSGENIC MOUSE WITH HbS PLUS HbS-ANTILLES
2421
occlusion of individual capillaries, one would expect to find
of cell-by-cell delay times, complete deoxygenation occurs
normal Purkinje cells next to those that have become pykbefore data collection begins at 2 milliseconds and hence
notic as is seen in Fig 6A. These findings are compatible
the oxygen affinity of the Hb will not affect the delay time.
with those found in some patients with sickle cell anemia
On the other hand, the solubility of the Hb will play a major
and related syndromes.
role because Eaton et all5 have shown that the delay time
Final manifestations of increased pathology are the reis proportional to the fractional saturation. The increased
duced birthrate and high level of spontaneous death of douexpression of human a chains will also contribute to enbly transgenic mice that are homozygous for the mouse pMajor
hanced polymer formation because the mouse a chain plays
deletion. The cause of early death was not clearly estabthe same inhibitory role toward polymerization in the
lished, but it appears to have multiple causes including contransgenic mouse as the human y chain in sickle cell anemia
gestion and repeated hepatic infarcts.
patients.' Hence, the short delay times can be attributed to
In a separate set of experiments on the exposed cremaster
the lower solubility of HbS-Antilles, which manifests itself
of living aHpsps-An'[pMDD]
mice, Kaul et a13' have reported
in the lower CsATdiscussed above and the higher expression
observing occasional sickled cells, RBC adhesion, and reof human a.
duced RBC velocity. This is the first time that in vivo RBC
Loss of urine-concentrating ability is a characteristic of
adhesion has been reported. The reduced RBC velocity when
human sickle cell patients that is found even in sickle trait
compared with control mice is compatible with increased
patients. The kidney is expected to be particularly sensitive
viscosity caused by in vivo polymer formation.
to sickle cell vasoocclusion because of the high osmolarity,
We conclude that the pS/pS-A"'
mice have a more severe
low pH, and relatively low oxygen tensions that are characphenotype than either parent line and that this increased
teristic of that organ. A urine-concentrating defect is not
severity is correlated with more extensive polymer formafound in aHpS[pMDD]
mice unless they are subjected to sevtion. Many of the pathologic features seen in the pS/pS-Ant
eral days of h y p ~ x i a We
. ~ found that urine-concentrating
mice under ambient conditions can only be elicited from the
ability was spontaneously decreased in both aH/3s/3S-A"'[pMD]
parent lines by placing them under hypoxic conditions,
and crHpSpS-"'[pMDD]
under ambient conditions. Because the
which also results in more extensive polymer formation.
percentage of PS and PS"""'of the aHPS/3S-Ant[pMD]
mice is
Because the pathology observed in the pS/pS-Ant
mice is similess than the percentage of PSin the parent crHps[/3MDD]
mice,
lar to that observed in the parent strain under hypoxic condithe reduced oxygen affinity of the S-Antilles Hb probably
tions, this is consistent with our earlier conclusion that the
plays a major role in the loss of urine-concentrating ability.
increased Rl3C density and renal concentrating defect seen
On the other hand, the loss of urine-concentrating ability
cannot be attributed to hypoxic renal damage in these aniin the aHpS[pMDD]
mice under hypoxic conditions was the
mals.
result of increased polymer formation and vasoocclusion
Organ damageto four of the doubly transgenic animals that caused by nondeformable RBCs. Comparing these mice to
werehomozygous for themouse pMaJor
deletion (aHflsps-h' the parent lines mayallow us to dissect out the features
of sickle cell disease that are most important in creating
[pMDD]
and were killed for experimentation was determined
histologically. Congestion was characteristic of all tissues.
pathology.
The liver showed numerous areas of both old and new focal
necrosis suggestive of episodic occurrence of ischemic
NOTEADDED IN PROOF
events. The observation that aspartate amino transferase and
At 10 days of age, aHpsps-Ant[fiMDD]
mice have a hematoalanine amino transferase levels were elevated in pSpS-Antcrit of 37 t 2 versus adult mice, which have a hematocrit
mice indicates that the process of liver infarction is an ongoof 16 5 2.5 (mean t SE, n = 3 and 16, respectively; P <
ing event in living animals. Focal necrosis of the liver has
.00002). Therefore, neonatal anemia is also a characteristic
been reported in both autopsy and biopsy specimens of sickle
of these mice.
cell patientsz6-" and has been attributed to vasoocclusive
events by some auth0rs.2~ The
spleen was enlarged more than
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