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1990 76: 745-754
Heritable severe combined anemia and thrombocytopenia in the
mouse: description of the disease and successful therapy
LL Peters, EC McFarland-Starr, BG Wood and JE Barker
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Heritable Severe Combined Anemia and Thrombocytopenia in the Mouse:
Description of the Disease and Successful Therapy
By Luanne L. Peters, Eleanor C. McFarland-Starr, Bonnie G. Wood, and Jane E. Barker
A new autosomal recessive mouse mutation, scat (severe
combined anemia and thrombocytopenia), causes intermittent episodes of severe bleeding in the homozygote. At
birth, affected mice are pale with intradermal petechiae
and bruises on exposed surfaces. Central nervous system
(CNS) bleeding occurs in 22% of the mice. Gastrointestinal
(GI) hemorrhaging and splenomegalyare noted in moribund
mice at autopsy. Of the 291 mice studied, 113 mice
survived the initial crisis and entered a spontaneousremission period lasting from day 16 to day 27. A second crisis
period ensued, and all but 22 mice died by 45 days. Mice in
crisis show significantly decreased platelets, erythrocytes,
and leukocytes and increased reticulocytes when compared to normal littermates. During remission all parame-
ters are significantly improved or revert to normal values.
Neither splenomegaly nor internal bleeding are observed
during remission. A platelet-specific antibody is present in
the plasma of mutant mice during crisis. The symptoms
(severe bleeding, anemia, low-platelet counts) and plateletspecific antibody production are transferred to lethally
irradiated normal mice through spleen cell transplantation.
Splenectomy of mice in remission significantly increases
survival. Exploitation of the features common to both
scat/scat mice and patients with some forms of autoimmune thrombocytopenic purpura will undoubtedly prove
useful in defining common pathways of disease development and in testing potential therapeutic measures.
0 1990 by The American Society of Hematology.
E
from autoimmune N Z B / B l N J - + / + ( N Z B ) mice were used as
positive controls during immunoblotting.
The Jackson Laboratory is fully accredited by the American
Association for Accreditation of Laboratory Animal Care.
Survival studies, gross pathology. and histopathology. The
average life span of the mutant animals and the temporal progression
of the disease state were determined by daily observations of 291
BALB-scatlscat mice for 60 days or until death occurred. The mice
were classified as being in crisis or in remission based on overt
symptoms. Mice in crisis were extremely pale, had bruised extremities, and occasionally had accumulation of blood in the dorsal cranial
area. Additional scat/scat mice not included in this study were killed
by cervical dislocation and autopsied as they became moribund.
Comparisons were made between mutant mice and age-related
BALB-+/ + mice killed at the same time. For histologic examination, tissues were fixed in Tellyznicksky’s fixative (thymus, lung,
lymph nodes, spleen, liver, kidney, and adrenal glands) or 10%
nonbuffered formalin (femur), embedded in paraffin, and sectioned
at 3 pm. Femurs were decalcified in Cal Ex (Scientific Products,
McGaw Park, IL). Sections were stained with Mayer’s hematoxylin
and eosin (H and E), periodic acid-Schiff reagent (PAS), methyl
green pyronin (MG/P), or Gomori’s iron stain.
Tissues for plastic sections were fixed in 0.3% glutaraldehyde0.5% paraformaldehyde in 0.05 mol/L PIPES buffer at pH 7.35,’O
Sections were stained with benzidene dihydrochloride.
XPERIMENTALLY produced immune platelet destruction in laboratory animals has been used to study
the effects of thrombocytopenia on megakaryocytopoiesis
and thrombop~iesis.’.~
While such models permit analysis of
in vivo platelet destruction and its consequences, they reveal
little about its etiology. Recently, a new recessive mutation
called scar (severe combined anemia and thrombocytopenia)
occurred in the BALBIcBy mouse strain. In homozygous
mice, presence of an antibody to a 110-Kd platelet protein
coincides with a cyclical platelet insufficiency. The disease,
including its immune component, is transferable through the
hematopoietic stem cells. In this regard the scat mutation
resembles known murine lupus models, including the NZB,
BXSB, and M R L strains, which are also transferable through
the stem cell^.^-^ In these strains, antiplatelet antibodies have
not been implicated in the pathogenesis of the respective
autoimmune syndromes. In scatlscat mice, however, antiplatelet antibodies appear to be a primary cause of the symptoms
produced by the gene defect.
The scatlscnt mice share a number of disease symptoms
and the presence of a platelet-specific antibody with patients
suffering from some forms of autoimmune thrombocytopenic
purpura (ITP). The current paper provides the first description of the defects in the mouse mutant, the transmission of
the disease through the hematopoietic stem cells, and the
development of one prophylactic measure.
MATERIALS AND METHODS
Animals. BALB/cBY (BALB)-scatlscat mice were discovered
and maintained at The Jackson Laboratory. Female mutant mice
were unable to survive pregnancy. As a consequence, the mutant
mice were generated either by mating BALB-scar/+ parents or by
transplanting ovaries from mutant females to congenic C.B. Hbb”
female mice and mating the ovary recipients to BALB-scat/ +
males.’A difference at the albino locus permitted recognition of mice
generated from the resident C.B.Hbb” oocytes and the transplanted
BALB oocytes. Progeny from the C.B.Hbb” oocytes were brown
agouti, while progeny from the BALB oocytes were albino. The
extreme pallor, bruises, and intradermal petechiae at birth easily
distinguished the scatlscat mice from their normal BALB littermates. Age-related BALB-+/+ mice generated from BALB-+/+
mating pairs were used as controls. Congenic C.B.Hbb5mice were
recipients in the transplantation studies. Erythrocytes and plasma
Blood, VOI 76, NO4 (August 15). 1990: pp 745-754
From the Jackson Laboratory, Bar Harbor, ME; and the Department of Zoology, University of Maine, Orono, ME.
Submitted November 3,1989; accepted April 26,1990.
Supported by a Graduate Student Board Research Grant from
the University of Maine (L.L.P.),a University of Maine Faculty
Research Grant (B.G.W.). and Grants No. DK 27726 (J.E.B.) and
HL 29305 (J.E.B.)from the National Institutes of Health.
The National Institutes of Health is not responsible for the
contents of this publication nor do the contents necessarily represent
the oficial views of that agency.
A preliminary report of this research has been published in
abstractform (Blood, 72:335a, 1988 [suppl 1. abstr 1250]).
Address reprint requests to Luanne L. Peters. PhD. The Jackson
Laboratory, 600 Main St, Bar Harbor, ME 04609-0800.
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.
6 I990 by The American Society of Hematology.
0006-4971/90/7604-O01 8$3.00/0
745
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746
Peripheral blood studies. Blood counts in mice are dependent
For this reason,
upon the method and time of blood
whole blood was withdrawn from the retro-orbital sinus between
1200 and 1400 hours. Erythrocytes were counted using a CoulterCounter Model ZBI. Leukocyte and platelets were counted by light
microscopy using conventional manual methods.I3 For differential
leukocyte counts, blood smears were prepared and stained with
Wright’s-Giemsa using an automatic Hemastainer (Geometric Data
Corporation). Differential counts were performed on 100 cells to
obtain the relative numbers (percent) of circulating lymphocytes and
granulocytes. Absolute counts were calculated by multiplying the
total white blood cell (WBC) count by the relative count. Reticulocyte percent was determined from direct counts of blood cells
exposed to new methylene blue.I4
Serum bilirubin determinations. Serum total-bilirubin levels
were determined by the Malloy-Evelyn method.I5
Urine screening. Qualitative urine hemoglobin and bilirubin
levels were determined using Ames Multistix 10 SG test strips.
Urine for microscopic analysis was obtained from the bladder using
a 30-gauge needle and examined by light microscopy using a 40x
objective for the presence of red blood cells (RBCs).
Indirect Coombs tests. RBCs from heparinized whole blood
obtained from the thoracic cavity of mice anesthetized with avertin
were washed four times and resuspended at 5% in normal saline
(0.9% NaCL in H,O). The cells were incubated 1:l with ficin
(Cooper Biomedical) for 10 minutes at 37OC, washed in saline, and
again resuspended to a 5% solution in saline. RBCs, autologous
plasma, and 22% albumin (Ortho) in a 1:2:2 ratio were incubated for
30 minutes at 37OC, centrifuged for 5 minutes at 138g, and read for
agglutination both macroscopically and microscopically.
Preparation of erythrocyte ghost membranes. Hemoglobin-free
erythrocyte membrane ghosts were prepared by the method of
Dodge et a1.I6 The amount of protein in the preparation was
determined by the method of Lowry.” The ghosts were dissolved in
4x Laemmli bufferla and boiled for 5 minutes before storage at
- 2OoC and/or before electrophoresis on polyacrylamide gels.
Preparation of solubilized platelet proteins. Platelets were
obtained from adult (6 to 8 weeks) BALB-+/+ mice. The mice
were anesthetized with avertin and blood from the thoracic cavity
was collected in 3.8% sodium citrate in saline. Plasma was removed
by centrifugation of the cells at 480g. The cells were washed two to
three times in saline to remove residual plasma. A platelet-rich
suspension in saline was obtained by a final centrifugation at 120g
for 20 minutes at 4OC. Platelets in the supernatant were adjusted to
2 x 108/mL and collected by centrifugation at 11,OOOg in a
Brinkman Eppendorf 5415 microcentrifuge. The pellets were washed
and vigorously vortexed in distilled water two times. The final pellet
was solubilized by addition of 50 pL of distilled H,O, 50 pL of 10%
sodium-dodecyl sulfate (SDS), and 50 pL of 4 x Laemmli buffer.
The solubilized platelets were boiled before storage at -2OOC
and/or before electrophoresis.
Electrophoresis on denaturing polyacrylamide gels. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell ghost
proteins and of solubilized platelet proteins was performed by the
method of Laemmli.I8Erythrocyte ghost proteins (125 pg) or 50 pL
of solubilized platelet proteins were loaded in each sample well.
Molecular weight standards (myosin [200,000 D], beta galactosidase [116,250 D], phosphorylase B [97,400 D], bovine serum
albumin [BSA, 66,200 D], and ovalbumin [42,699 D]) were applied
to one lane as markers. Electrophoresis was continued for 5 hours at
a constant current of 40 mA or overnight at 8 mA. The proteins were
either stained with Coomassie blue’’ or transferred to nitrocellulose
for Western analyses.20,21
Western blot analyses. Heparinized whole blood was obtained
PETERS ET AL
from the thoracic cavity under avertin anesthesia. Untreated plasma
or plasma precipitated with antimouse IgG (Sigma, St. Louis, MO;
M8890) overnight at 4°C was tested for erythrocyte and platelet
autoantibodies. A nitrocellulose filter with red-cell or plateletmembrane proteins was placed in buffered saline (20 mmol/L TRIS,
500 mmol/L NaCl, pH 7.5) for 20 minutes and blocked in 3%
gelatin in buffered saline with 0.05% Tween-20 for 1 hour at room
temperature. The filter was rinsed in buffered saline containing
0.05%Tween-20 and incubated for 2% hours in serum diluted 150 to
1:lOO in 1% gelatin in Tween-20 buffered saline. The filter was
washed two times in Tween-20 buffered saline, incubated with goat
antimouse IgG (heavy and light chains) horseradish peroxidase
conjugate (Bio-Rad, Richmond, CA) for 1 hour, washed and
immersed in 0.3% horseradish peroxidase color development reagent
(4-chloro-1-naphthol, Bio-Rad) in cold methanol with 0.02% H202
in buffered saline for 5 to 30 minutes.
Bone marrow transplantation. At least 2 x lo6 spleen cells
retrieved from mutant mice in crisis were transplanted intravenously
(IV) into C.B.Hbbs mice with an LD” dose (700 R) delivered from
a ”?Cs gamma irradiator at a dose rate of 220 rads/min.*, The
difference at the beta globin locus, Hbb, allowed measurement of
donor cell contribution to the host. Hemoglobin phenotype was
determined on blood lysates by the technique of W h i t n e ~ Blood
.~~
and plasma were obtained from moribund mice for the various
analyses.
Splenectomy. Animals were splenectomized using standard
technique^.^^
Statistical analyses. Numerical results were analyzed by Tukey’s
test for significance of difference between mean values and analysis
of variance (ANOVA) or the two-tailed student’s t test.25All data
expressed as percentages were transformed prior to statistical
analysis. Survival studies were analyzed by the xz test.26
RESULTS
Progeny testing. The scat mutation was inherited in a
recessive fashion. Breeding of known heterozygotes, derived
from matings of C.B.Hbb”females carrying scatlscat ovaries
to BALB- / males, resulted in the appearance of affected
newborns. Crosses between BALB-scat/ and BALB- /
mice never produced affected offspring. In heterozygous
matings, approximately 18% of live offspring were affected,
suggesting some death in utero occurred. The chromosomal
location of the scat mutation has not yet been determined.
Survival studies and gross pathology. Newborn BALBscatlscat mice were monitored daily for a period of 60 days or
until death occurred (Fig 1). All the homozygous mice were
pale at birth and showed bruising and petechiae. Twenty-two
percent of the mice developed intracranial bleeding. Of the
291 mice studied, 178 (61.2%) died by 25 days without
entering a remission stage. The remaining mice went into a
spontaneous remission period in which all overt symptoms
had completely disappeared by day 16. On average, this
remission period lasted 11 days, ending a t day 27. During the
second crisis, 91 mice (31.3%) died while 22 (7.5%) recovered and entered a n extended remission period free of all
symptoms. These animals were not monitored more than 60
days. Of the mutant mice studied, 90% were dead by 50 days,
with a mean survival time of 22 days. T h e mean life span of
BALB-+/ + mice is greater than 18 months.27
Gross pathology a t autopsy revealed extensive gastrointestinal (GI) and rectal bleeding. There was marked spleno-
+ +
+
++
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747
HERITABLE ANEMIA AND THROMBOCYTOPENIA IN MICE
L
3
aI
8
*OI
0
10
20
30
40
50
60
70
age (days)
Fig 1. The survival of swr/scar mice as a function of age. Data
is based on the daily observation of 291 animals.
megaly (Table 1). Mice killed during remission periods
showed no internal bleeding and the spleen size was normal.
Thymus, kidney, lung, lymph node, and adrenal glands
from scatlscat mice in crisis appeared histologically normal.
Hemosiderin deposits were not observed in mutant animal
tissues during crisis episodes using Gomori's iron stain.
Spleens of scatlscat mice in crisis showed increased mature
megakaryocytes and a proliferation of large mononuclear
cells that completely effaced the normal splenic architecture
(Fig 2A through C). Lymphatic nodules and blood sinuses
were conspicuously reduced or absent. The mononuclear cells
were characterized by a large, pale nucleus with dispersed
chromatin and a small amount of cytoplasm (Fig 2D through
F). Mitotic figures were frequent. Spleens of scatlscat
animals in remission were histologically normal. Benzidine
positive cells were absent or present only in small numbers in
scatlscat crisis spleens, whereas a strong reaction was evident, as expected, in the red pulp areas of +/ + and scat/scat
remission spleens. No proliferation of plasma cells or Mott
cells could be found using MG/P or PAS staining and
conventional morphological criteria in any of the sections
studied.28
Animals in crisis showed foci of mononuclear cells in the
perivascular areas of the liver (Fig 2G). Mitotic figures were
commonly seen in these foci. Whether these cells are derived
from or are similar to those of the spleen is unknown at
present. No other organs showed any cellular infiltrates such
as those found in the liver. Liver sections from animals in
remission did not differ histologically from + / + animals.
Examination of femurs showed increased megakaryocytes
in scatlscat mice in crisis when compared to either + / + or
scat/scat mice in remission (Fig 2H and I). The cellularity
appeared normal in most cases, although moribund animals
frequently showed increased fat deposition in the marrow.
Peripheral blood. The extreme pallor of the mice in
crisis was indicative of a severe anemia. Anisocytotic and
poikilocytotic erythrocytes were noted in blood smears from
crisis mice, and there were frequent burr cells, schistocytes,
and acanthocytes, and markedly increased numbers of poly-
chromatophilic erythrocytes (Fig 25 through L). RBC counts
from 17 mice in crisis at 20 to 26 days postnatally averaged
2.62 x 10l2/1 2 0.24 x 10"/1 (mean f SEM). Countsfrom
16 normal-appearing littermates averaged 8.88 x 10l2/1
0.29 x 10L2/1.Hematocrits from 34 mice in crisis were
15.8% f 1.3%, while those from 8 mice in remission were
41.4% f 2.6% and were similar to the mean value of 42.6% f
0.9% from 32 normal mice. The absolute reticulocyte count
was increased from 0.81 x 10t2/Lf 0.08 x 10l2/L in normal
mice (n = 12) to 1.97 x 10I2/L + 0.15 x 10'2/Linscat/scat
mice in crisis (n = 8).
f i e intradermal petechiae, bruising, and anemia noted at
birth and during crisis periods were suggestive of a severe
bleeding disorder. Bleeding times were noted to be increased
in scatlscat mice during routine retro-orbital bleeding or
when obtaining blood from tail veins. Clotting in both cases
occurred almost immediately (less than 5 minutes) in BALB+ / + mice, whereas BALB-scat/scat mice required cauterization of tail veins. In the case of retro-orbital bleeding,
BALB-scatlscat mice usually bled to death. Subsequent to
this observation, retro-orbital bleeding, when performed in
BALB-scatlscat mice, was immediately followed by euthanasia. Prolongation of bleeding time suggested a platelet
deficiency. The mutant mice were severely thrombocytopenic
during crisis periods (Fig 3). During remission periods,
complete (males) or near-complete (females) recovery occurred. On blood smears there was no evidence of platelets in
crisis mice, although platelets were present and appeared
normal in mice in remission (Fig 2K and L).
Total WBC counts indicated that crisis mice were severely
leukopenic (Table 2). During remission periods, female
WBC counts did not differ from normal controls, while male
WBC counts improved but remained significantly (P< .OS)
less than that in normal males. Differential counts of blood
smears showed an increase in the percentage of granulocytes
and a decrease in the percentage of lymphocytes. Absolute
counts based on these values indicated a severe lymphopenia
that persisted in remission males but not females. Monocytes
and eosinophils consistently represented 1% to 5% of the
peripheral blood white cells and did not differ among the
mice studied.
Serum bilirubin levels and qualitative urine screening.
Serum bilirubin levels did not significantly differ between
BALB-scatlscat mice in crisis and BALB- + / + controls. For
2- to 4-week-old scat/scat mice in crisis, total serum bilirubin
levels were 13.2 pmol/L f 1.7 pmol/L (n = 9) compared to
10.1 pmol/L f 1.7 pmol/L (n = 5) for +/+ mice of the
same age. Qualitative urine screening showed no increase in
urinary bilirubin in scatlscat crisis mice. Increased urine
Table 1. Spleen Weight
Genotype
Phenotype
No. Mice
+I+
Normal
Crisis
Remission
33
scat/scar
scat/scat
14
12
Values are mean i SEM.
* P< .05 versus normal and remissionanimals.
Weight of Spleen
I% body weight)
0.65 i 0.05
3.00 & 0.20.
0.60 i 0 . 1 4
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748
Pl3ERSlTM
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HERITABLE ANEMIA AND THROMBOCYTOPENIA IN MICE
Uj
B
males
0 females
(25)
+ +
Fig 3. Platelet (PLT) counts in normal ( / 1 and scat/scat
mice in crisis (Cr) and in remission (Rem). Numbers in parentheses
indicate the number of mice analyzed; bars indicate the SEM.
.indicates a P <.05 when scat/scat mice in remission are compared with / mice; **, a P <.05 when scat/scat mice in crisis
are compared with both mutant mice in remission and / mice.
+ +
+ +
hemoglobin could be detected using Ames Multistix reagent
strips; scatlscat mice in crisis frequently gave 3+ reactions,
while normal mice showed a trace a t most. When urine was
examined under the microscope, intact RBCs were abundant
(greater than 20/hpf) in scatlscat crisis but not +/ + mice.
Examination of serum for autoantibodies. In six of nine
trials, plasma from mice in crisis caused a diminution of
platelet numbers when injected into the tail veins of BALB+/+ mice (Fig 4). At 90 minutes postinjection, platelet
counts ranged from 62% to 83%of baseline values. N o effect
was seen in any of six recipients of control (BALB-+/+)
plasma. N o effect on erythrocyte morphology was detected in
any of the recipients. Indirect Coombs' tests were negative
for mice in crisis (n = 6), further suggesting that the effect
of plasma injection was limited to platelets. By indirect
Coombs' testing, erythrocyte autoantibody was easily detected in aged N Z B (n = 3) positive controls.
To determine whether the platelet inhibitory factor was an
autoantibody, plasma from BALB- + / +, scatlscat mice in
crisis and in remission, and positive control plasma from the
autoimmune N Z B mice was reacted with erythrocyte membrane and/or platelet proteins affixed to nitrocellulose. All
plasma samples were tested against autologous erythrocyte
ghost proteins. In eight trials no circulating erythrocyte
autoantibodies were detected in plasma from the mutant
mice or from BALB-+/+ mice (Fig 5). Plasma from
autoimmune N Z B mice gave strong positive reactions to
749
NZB, BALB-scatlscat and - + / + erythrocyte membrane
proteins.
For detection of platelet-specific antibodies, it was necessary to react both BALB-+/ + and scat/scat plasma against
BALB- + / + platelet proteins, since insufficient scatlscat
platelets were available for platelet membrane preparations
and electrophoresis. Our assumption was that a plateletspecific antibody, if present in scatlscat mice, would cross
react with normal platelet proteins. A specific BALB- + / +
platelet membrane protein gave a strong positive reaction
with plasma from 12 of 14 scatlscat mice in crisis (Fig 5). In
each case the platelet protein had an apparent mol wt of 110
Kd. There was no reactivity with the 110-Kd protein when
the filters were probed with plasma from BALB- + / + mice
(n = 1 1). Of five scat/scat remission mice tested, one showed
a weak positive reaction and four were negative. The probability that the antibody is an IgG moiety is enhanced by the
observation that scatlscat crisis plasma precipitated with
anti-IgG no longer recognizes the 1 IO-Kd platelet protein.
Disease transmittance through spleen cell transplantation. To determine whether the disease is intrinsic to
hematopoietic progenitors, spleen cells from mice in crisis or
from normal mice were transplanted into lethally irradiated
+ / + C.B.Hbb" congenic hosts. All the recipients assumed
the complete hemoglobin phenotype of the mutant mice 3 to
8 weeks after transplantation. The recipients of scat/scat
cells but not + / + cells all displayed overt symptoms of the
scat gene defect in an episodic manner within 8 weeks.
Animals were autopsied as they became moribund (3 to 11
weeks post-transplantation). When compared to mice receiving + / + cells (autopsied at 8 to 11 weeks post-transplantation), mice receiving scatlscat cells had significantly increased spleen size and significantly decreased peripheral
blood counts that exactly paralleled the changes seen in
scatlscat crisis versus + / + animals (Table 3). In addition,
the spleen and liver histology and RBC morphology were
altered such that they took on the scatlscat crisis appearance
(Fig 6A through C). N o change in spleen histology or
erythrocyte morphology was seen in mice receiving +/+
cells.
Western analyses detected antibody to the 1IO-Kd platelet
protein in 60% of the scatlscat recipients (not shown). None
of the mice receiving +/ + cells demonstrated any antiplatelet antibody.
The results indicated that the disease was transmitted by
mutant spleen cells into irradiated +/ + congenic hosts.
Effects of splenectomy on disease genesis. The splenomegaly and autoimmune aspects of the disease in scatlscat
4
Fig 2. Microscopic appearance of tissues in normal and mutant mice. (Original magnification indicated in parentheses.) (A) Spleen of a
scaf/scatcrisis mouse ( x 100). (6) Spleen of a scadscat remission mouse ( x 100). ( C )Normal spleen ( x 100). The scat/scatcrisis spleen no
longer shows lymphatic nodules and has increased numbers of megakaryocytes. (D and E) Enlargements of spleen sections of scar/scat
crisis mice showing the large, proliferating mononuclear cells that efface the normal splenic architecture ( x 1.OOOl. (F) Section of a normal
spleen through the center of a lymphatic nodule ( x 1,OOO). Note the smaller size of the cells compared with the scaf/scaf crisis spleen. (G)
Perivascular focus of cells in the liver of a scaf/scat crisis animal (~400).
A group of cells of unknown origin is located immediately left of a
liver microcapillary. (H) Femur from a scaf/scafmouse (~400).
(1) Normal femur (~400).
Note the increased number of megakaryocytesin
the scaf/scat crisis femur. (J) Peripheral blood smear taken from a normal mouse (x1,OW). (K) Peripheral blood smear taken from a
scadscat mouse in remission (x1,OOO). (L)Peripheral blood of a scadscat crisis mouse ( x 1,OOO). The erythrocyte morphology is grossly
abnormal and platelets are completely lacking in the scat/scat crisis mouse.
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750
PETERS ET AL
Table 2. WBC Counts in Normal and scat/scat Mice in Crisis and in Remission
Group (No.)
Absolute
Granulocyte
Count
wBC Count (x~o’/L)
Granulocytes (%)
13.8 f 0.5
15.5 f 1.3
2.2
12.5 f 2.1
4.2 & 4.0t
15.2 f 2.1
43.4 f 4.0t
11.6 f 0.3
LvmDhocvtes (%)
Absolute
Lymphocyte
Count
(X1 0 ~ ~ 1
Females
+/+ (291
scat/scat
Rem (5)
Cr (13)
* 0.2
82.2
1.8 0.3
2.2 f 0.8
*
82.4 f 2.8
53.8 4 . l t
18.7 f 1.4
2.2 f 0.2
79.1 f 1.5
9.3
20.9
44.6
1.7 f 0.2
1.5 f 0.2
70.2 f 3.2
50.6 f 7.1 t
5.7 f 0.6+
2.0 f 0 . 5 t
f
1.4
11.4 f 0.4
10.5 k 1.9
2.3 f 0.4t
Males
+/+ (25)
scat/scat
Rem ( 8 )
Cr ( 8 )
8.1 f 0.6+
3.4 f 0.5t
f
f
1.6
7.7t
0.3
Values are mean f SEM.
Abbreviations: Cr, crisis; Rem, remission.
* P < .05 versus +/+.
t P < .05 versus +/+ and scat/scat Rem.
mice in crisis suggested that splenectomy might be a therapeutic measure. Splenectomy was performed in 14 mutant
animals. All animals were in remission at the time of surgery
as judged by overt symptoms and peripheral blood WBC and
PLT counts. Twelve of the mice (86%) survived at least 60
days without any overt indications of disease. Five healthy
mice with no sign of bruises or petechiae were killed a t 60
days. Before death, peripheral blood counts revealed normal
hematocrits and platelet counts, normal WBC counts, normal differential counts and normal reticulocyte counts (Table 4). Of those mice remaining, all survived for longer than
10 months without ever showing scat symptoms.
0
I
I
I
30
60
90
Time (minutes)
+ +
Fig 4. Platelet (PLT) counts (&SEMIin normal / mice after
or scat/scat Cr plasma
injection with 0.1 cc / plasma 1-(
(x-x).
Only those animals that responded
the injection of
scat/sc8t Cr plasma (six of nine) are shown.
+ +
The survival data (12 of 14 splenectomized mutant mice
survived in remission for at least 60 days compared to 22 of
291 unperturbated mutant mice that had survived in remission for 60 days) indicated that splenectomy significantly
(P< .005) improved survival of the mutant mice.
Screening of normal littermates. Unaffected offspring
(scat/?)of scat/ + mating pairs were overtly normal; scat/ +
(50%) pups could not be distinguished from + / + (25%)
pups a t birth or subsequently. To determine if scat/+
littermates could be identified by other parameters, 17
normal-appearing littermates taken from litters in which at
least one affected animal was produced were compared to
age-related + / + mice born of BALB- + / + mating pairs.
The normal-appearing littermates did not differ from + / +
offspring of + / + parents in WBC, platelet, reticulocyte, or
differential counts or in erythrocyte morphology or hematocrit values (data not shown). Also, two populations of
normal littermates representing scat/ +, and + / + genotypes were not evident. In terms of spleen weights, however,
offspring of scat/+ mating pairs had consistently and
significantly (P< .05) increased spleen sizes compared to
offspring of + / + mating pairs. The spleen weight, as percent
body weight, was 1.02 0.03 for scat/? mice and 0.73 f
0.03 for + / + mice. The absolute spleen weight was also
significantly (P< .05) elevated in scat/? versus + / + mice
(data not shown). Histologically no abnormalities in the
spleens of scat/? animals could be identified by light microscopy, indicating that the changes at this stage were subtle.
Western analysis for platelet antibody was negative in all
17 normal appearing offspring of scat/ + mating pairs.
DISCUSSION
The results presented here indicate that scatlscat mice
suffer from an immune disorder. The presence of an antibody
specific for a 110-Kd platelet protein in mice that are platelet
deficient suggests cause and effect. The platelet insufficiency
is manifested in low platelet counts and a lack of platelets on
peripheral blood smears. (Manual platelet counts are falsely
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
HERITAELE ANEMIA AND THROMEOCnOPENIA IN MICE
76 1
Fb5. W m t u n b k t ( i n g t e d . N c t ~ o u toombadin. Nltrocdh~k..likmconutning /
(lono 1) ond scmt/.ur (hno2) orvthrocytoghoots
protoins w u o probod with plomu from an NZE
mouu (NZEI. plowno from a scmt/scmt mouu in
aisir ( L u r Cr). or plouru from a scmt/scmt m o u ~
in romiu&n [#car Run). Nitrocolluk.. fikrr contoining plotdot protdnr from + / + mko (lonos 3
through 6) w u o pra4ul with plouru from 0 #cat/
scmt mouu in a h i r ( . u t Cr). plowno from 0
scmt/scmt mouoo in rominion ( . u t Run). or plouru
from 0 + / + nwn~80( + / + 1 . Tho posttivo bond
s p o tho
~ width ol tho wdl in tho . u r / . u t Cr hno
ond is opproxinutoty 110 Kd.
+ +
afTected mice are frequently weanling. This suggests antibody is produced in situ rather than transferred from the
mother at thisstage.
Autoantibodies to specific platelet membrane glycoproteins (GP) are present in some humans with autoimmune
thrombocytopenic purpura.” In many cascs such antibodies
appear to be directed against human GP Ib and GP Ilb/
llla.”’ In the present study antibodies to the as yet
unidentified I IO-Kd platelet membrane protein were detected in the plasma of I2 of 14 scut/scu~mice. Plasma from
normal mice was consistently negative, and only one mouse
classified as being in remission displayed reactivity. Initial
tests showed that nonspecific protein binding occurs at
plasma dilutions of 5 1:40 in all groups (data not shown).
Hence all samples were diluted at least 150 in this study.
Similar problems in obtaining low background using human
material have been encountered with both serologic and
immunoblot methods.’4.” In addition. as was the case with
two scut/scur mice tested by immunoblotting and three tested
by serum injections. circulating antiplatelet antibody is not
always detectable in humans with ITP using either Western
analysis or other methods.’O.Y”
elevated by the presence of many microerythrocytes and
unlysed erythrocytes with numerous membrane spicules.)
The onset of the disease at birth is difficult to explain. since
the immune system of the newborn is not yet fully developed.
Recently we investigated the possibility that maternal antibodies are involved in the etiology of the defect. Plasma from
pregnant scar/ + but not from virgin scur/ + mice is, in fact,
positive for platelet antibody by Western blotting (paper in
preparation). The etiology is suggestive of a platelet-limited
transplacental-mediated disease in the scur/scur mice. The
initial crisis in scur/scuf newborn mice may be due to
maternal antibody crossing the placenta.
The observation that spleen weights are increased in
normal-appearing littermates of scur/scur mice further s u p
ports the hypothesis of a maternally mediated efTect. Our
Western blot results and plasma injection data clearly show
that the antibody present in scur/scur crisis mice crossreacts
with normal platelets. Hence. one could predict that the
increased spleen size in normal littermates (apparently
regardless of genotype) represented a response to an antibody that was present in utero and then cleared after birth.
The second crisis period is not so easily reconciled. since the
-
T.bk3.80k.n(UI..ndP~lBkod~.tAutopryk,+/+C.I).~ConOMio~A*.rL.rtullmdinionuwl
A.popu(nknwith8c8r/.crtor
+/+ 8pk.nC.lh
bb”
U T m
Injmd(N0.)
Bp*mwJghc
pM**cQm
IXBodvWadw) H m ” l % l
(xl0’’A)
48.2 t 0.7
30.0 i3.4.
1.35 t 0.04
+/+ (3)
0.43 I 0.02
W s w r ( 9 ) 1.41 t 0.43.
=
V d u e r m m ~ SEM.
.PC .06MnrP + I + .
0.034 I 0.006.
WBCcQm
(xlO*A)
*
14.6 1.4
9.4 t 2.4.
*kolua
LP@=V-
cimJaVarl’16) bunt(xlO*A) LymphocYllI) buniIxlO’A1
14.3
46.3
2
2.0
- 5.1.
*
2.1 0.4
4.0 I 1.3
84.3 t 2.3
49.6 t 6.2.
12.3 t 1.2
5.0 t 1.3.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
PETERS ET AI.
762
Fig& ~ ~ a ( . p k . n . n d b k n n l t r o m o n o l g l . n t o t m u t ~ t a e I t ax .l 2d m 7 a r / m 7 a r C r r p k . r , ~ m r o I n ~
Into a / C.B.Hb6. motno. R . d p k m th.u# -0
r “ d
8 W o hr.Photomkrographa(wigid-)
aro of (A) .pk.n
(xloo): (E) qJl0.n ~xl.OO0);and (C) p0riph.r.l Mood l xl.OO0).
+ +
Antibodies to erythrocytes have not been detected either
by immunoblotting or by the indirect Coombs’ test. During
crisis episodes mice were not visibly jaundiced. Serum and
urine bilirubin values did not differ from controls. In addition. no evidence of increased intravascular RBC destruction
in kidney was Seen using Gomori’s iron stain. These observations suggest the anemia in the scur/scur mice in crisis is a
consequence of bleeding due to the platelet insufficiency. The
extreme pallor of newborn mice may result from excessive
bleeding at birth. The development of misshapen erythrocytes as the mice become moribund is more difficult to
understand. The scur/scur mice in crisis have grossly enlarged spleens. Unlike the human spleen, however. the mouse
spleen functions as an adjunct hematopoietic organ throughout life.’* Splenomegaly in scur/.wur crisis mice could,
therefore. be an indicator of compensatory erythropoiesis.
However, if this were the case. splenectomy would be
expected to worsen the anemia, not cure it. Furthermore. the
histopathology of the spleen is not consistent with increased
RBC proliferation but instead shows large mononuclear cells
of unknown function. Based upon morphological characteristics. MG/P, PAS, and benzidine staining. these cells are
neither erythrocyte precursors nor plasma cells. Misshapen
erythrocytes may be a consequence of the altered role of the
spleen in crisis mice. Further studies to characterize the
spleen cell population in scur/scur mice are underway.
The primary cause of the murine disease is not clear. It is
possible that the disease symptoms in the scur/scur mice are
related loa spleen-limited neopla~m.”.~
It is noteworthy that
such neoplasms in human beings are characterized by a lack
of lymph node involvement. but liver involvement is often
noted. Autoimmune complications of both platelets and
erythrocytes occur secondary to myeloproliferativeand lymphoproliferative disorders in human being^."^' The destruction of splenic architecture and accumulation of large mononuclear cells in scur/scur mice during crisis are consistent
with a neoplasticsyndrome. Should such a disease exist in the
mouse, one would predict the disease would be transferrable
through hematopoietic “stem” cells. The fact that the
syndrome is acquired by normal recipients of mutant spleen
cells is consistent with the transfer of the following cell type:
a spleen-derived neoplastic cell; an antibody-producingcell
capable of proliferating in the host; or a cell racogniable as
foreign by both host and donor.
The spleen appears to be critical to the genesis of the
sccond crisis. since splenectomy during the initial remission
halts disease progression. One can hypothesize that splenectomy cures the disease in scur/scur mice. as in human being
with ITP, by removing a site of antiplatelet antibody synthesis and/or of platelet destruction.16*’
There are a number of similarities between the murine
disease and idiopathic thrombocytopenic purpura (ITP) in
human beings. These include IgG-platelet antibody. mucosal
bleeding. dermal bleeding (petechiae), increased numbers of
megakaryocytes. and intracranial hemorrhaging.’“”’ However. the resemblance is not absolute. ITP in human beings is
Tabb4. P u i p h u o l B k o d c . ( h h ~ # n / # n M k o
WBccM1
p*a*ccM1
( x 1O’AI
Ix lO”A1
eanb2“(%I
20.8 t 2.9
1.24 t 0.2
23.4 t 3.9
LVmOhocVtr1%)
74.4
f
4.4
l+“ah(%I
46.0
= 0.3
Velum au “
I
2 SEM. N o d i f f a ” wen mtod b
”msb..ndfa”. .ndt h e ~ ~ p o o han
b dfive mice.
R.clcJocyar (%I
1.3 t 0.3
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
753
HERITABLE ANEMIA AND THROMBOCYTOPENIA IN MICE
not associated with significant splenomegaly, misshapen
erythrocytes, or leukopenia.50 An important point is that both
diseases are characterized by circulating antiplatelet antibodies and diminution of platelet number. This means that
exploration of the murine disease should uncover common
developmental pathways, consequences, and prophylactic
measures for platelet insufficiency diseases.
ACKNOWLEDGMENT
We thank Kelly Edwards and Stan Short for expert photographic
services, Ann Higgins and Priscilla Jewett for preparing slides, Drs
Edward Birkenmeier and Leonard Shultz for helpful comments on
the manuscript, Dr Janan Eppig for assistance with the figures, and
Barbara Dillon for secretarial services.
REFERENCES
1. Ebbe S, Yee T, Carpenter D, Phalen E Megakaryocytes
increase in size within ploidy groups in response to the stimulus of
thrombocytopenia. Exp Hematol 1655,1988
2. Ebbe S, Levin J, Miller K, Yee T, Levin F, Phalen E:
Thrombocytopoietic response to immunothrombocytopenia in nude
mice. Blood 69:192, 1987
3. Corash L, Chen Y, Levin J, Baker G, Lu H, Mok Y
Regulation of thrombopoiesis:Effects of the degree of thrombocytopenia on megakaryocyte ploidy and platelet volume. Blood 70:177,
1987
4. Jyonouchi H, Kincade PW, Good RA, Fernandes G: Reciprocal transfer of abnormalities in clonable B lymphocytes and myeloid
progenitors between NZB and DBA/2 mice. J Immunol 127:1232,
1981
5. Morton JI, Siege1 BV: Transplantation of autoimmune potential. I. Development of antinuclear antibodies in H-2 histocompatible recipients of bone marrow from New Zealand Black mice. Proc
Natl Acad Sci USA 71:2162,1974
6. Eisenberg RA, Izui S, McConahey PJ, Hang L, Peters CJ,
Theofilopoulos AN, Dixon FJ: Male determined accelerated autoimmune disease in BXSB mice: Transfer by bone marrow and spleen
cells. J Immunol 125:1032, 1980
7. Theofilopoulos AN, Balderas RS, Gozes Y, Aguado MT, Hang
L, Morrow PR, Dixon FJ: Association of Ipr gene with graft-vs.-host
disease-likesyndrome. J Exp Med 162:1, 1985
8. Theofilopoulos AN, Koftler R, Singer PA, Dixon FJ: Molecular genetics of murine lupus models. Adv Immunol46:61, 1989
9. Robertson GG: Ovarian transplantation in the house mouse.
Proc Soc Exp Med 44:302,1940
10. Baur PS, Stacey TR: The use of PIPES buffer in the fixation
of mammalian and marine tissues for electron microscopy. J
Microscop 109:315, 1977
11. Russell ES, Bernstein SE: Blood and blood formation, in
Green EL (ed): Biology of the Laboratory Mouse, 2nd ed. New
York, NY, McGraw-Hill, 1968, p 351
12. Clark RH, Korst DR: Circadian periodicity of bone marrow
mitotic activity and reticulocyte counts in rats and mice. Science
166:236, 1969
13. Brown BA: Hematology: Principles and Practices, 4th ed.
Philadelphia, PA, Lea and Febiger, 1984, p 59
14. Deiss A, Kurth D: Circulating reticulocytes in normal adults
as determined by the new methylene blue method. Am J Clin Pathol
53:481, 1970
15. Bauer JD: Clinical Laboratory Methods, 9th ed. St Louis,
MO, Mosby, 1982, p 538
16. Dodge JT, Mitchell C, Hanahan DJ: The preparation and
chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100:119, 1963
17. Lowry OH, Rosenberg NJ, Farr AL, Randall RJ: Protein
measurement with folin phenol reagent. J Biol Chem 193:265, 1951
18. Laemmli UK: Cleavage of the structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680, 1970
19. Bodine DM IV, Birkenmeier CS, Barker JE: Spectrin deficient inherited hemolytic anemias in the mouse: characterization by
spectrin synthesis and mRNA activity in reticulocytes. Cell 37:721,
1984
20. Burnette WN: Western blotting: Electrophoretic transfer of
proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and
radioiodinated protein A. Anal Biochem 112:195, 1981
21. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure
and some applications. Proc Natl Acad Sci USA 76:4350,1979
22. Lewis JP, OGrady LF, Bernstein SE, Russell ES, Trobaugh
SE Jr: Growth and differentiation of transplanted W / W marrow.
Blood 30601,1967
23. Whitney JB 111: Simplified typing of mouse hemoglobin
(Hbb)using cystamine. Biochem Genet 16:667,1978
24. Haller JA Jr: The effect of neonatal splenectomy on mortality
from runt disease in mice. Transplantation 2:287, 1964
25. Guenther W C Analysis of Variance. Englewood Cliffs, NJ,
Prentice-Hall, 1964, p 43
26. Schefler WC: Statistics for the Biological Sciences, 2nd ed.
Reading, MA, Addison-Wesley, 1980, p 103
27. Frith CH, Suber RL, Umholtz R: Hematologic and clinical
chemistry findings in control BALB/c and C57BL/6 mice. Lab
Animal Sci 30835,1980
28. Shultz LD, Coman DR, Lyons BL, Sidman CL, Taylor S:
Development of plasmacytoid cells with Russell Bodies in autoimmune “viable motheaten” mice. Am J Pathol 127:38, 1987
29. Rock G, Decary F, Tittley P, Fuller V: Electroblotting and
immunohistochemical staining for identification of platelet antibodies. Br J Haematol67:437, 1987
30. McMillan R, Tani P, Millard F, Berchtold P, Renshaw L,
Woods VL Jr: Platelet-associated and plasma anti-glycoprotein
autoantibodies in chronic ITP. Blood 70:1040, 1987
3 1. Devine DV, Currie MS, Rosse WR, Greenberg CS: PseudoBernard-Soulier syndrome: thrombocytopenia caused by autoantibody to platelet glycoprotein Ib. Blood 70:428, 1987
32. Szatkowski NS, Kunicki TJ, Aster RH: Identification of
glycoprotein Ib as a target for autoantibody in idiopathic (autoimmune) thrombocytopenic purpura. Blood 67:310, 1986
33. Tomiyama Y, Kurata Y, Mizutani H, Kanakura Y, Tsubakio
T, Yonizawa T, Tarui S:Platelet glycoprotein IIb as a target antigen
in two patients with chronic idiopathic thrombocytopenic purpura.
Br J Haematol66:535,1987
34. Tsubakio T, Tani P, Woods VL, McMillan R: Autoantibodies
against platelet GPIIb/IIIa in chronic ITP react with different
epitopes. Br J Haematol67:345, 1987
35. Menitove JE, Pereira J, Hoffman R, Anderson T, Fried W,
Aster RH: Cyclic thrombocytopenia of apparent autoimmune etiology. Blood 73:1561, 1989
36. Cines DB, Schreiber AD: Immune thrombocytopenia. Use of
a Coombs antiglobulin test to detect IgG and C3 on platelets. N Engl
J Med 300:106,1979
37. Hedge UM, Powell DK, Bowes A, Gordon-Smith EC: Enzyme linked immunoassay for the detection of platelet associated
IgG. Br J Haematol48:39, 1981
38. Hummel KP, Richardson RL, Fekete E: Anatomy, in Green
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
754
EL (ed): Biology of the Laboratory Mouse, 2nd ed. New York, NY,
McGraw-Hill, 1968, p 247
39. Kehoe J, Straus DJ: Primary lymphoma of the spleen.
Clinical features and outcome after splenectomy. Cancer 64: 1433,
1988
40. Andouin J, Diebold J, Schvartz H, Le Tourneau A, Bernadou
A, Zittoun R: Malignant lymphoplasmacytic lymphoma with prominent splenomegaly (primary lymphoma of the spleen). J Pathol
155:17, 1988
41. McMillan R, Longmire RL, Yelenosky R, Smith RS, Craddock CG: Immunoglobulin synthesis in vitro by splenic tissue in
idiopathic thrombocytopenic purpura. N Engl J Med 286:681, 1972
42. Duhrsen U, Augener W, Zwingers T, Brittinger G: Spectrum
and frequency of autoimmune derangements in lymphoproliferative
disorders: Analysis of 637 cases and comparison with myeloproliferative disease. Br J Haematol64235, 1987
43. Aghai E, Quitt M, Lurie M, Anta1 S, Cohen L, Bitterman H,
From P: Primary hepatic lymphoma presenting as symptomatic
immune thrombocytopenic purpura. Cancer 60:2308,1987
44. Schwartz KA, Slichter SJ, Harker LA: Immune-mediated
platelet destruction and thrombocytopenia in patients with solid
tumors. Br J Haematol51:17, 1982
45. Verdirame JD, Feagler JR, Commers JR: Multiple myeloma
associated with immune thrombocytopenic purpura. Cancer 56:
1199,1985
PETERS ET AL
46. Ballen PJ,Segal GM, Stratton JR, Gernsheimer T, Adamson
JW, Slichter SJ: Mechanisms of thrombocytopenia in chronic
autoimmune thrombocytopenic purpura. Evidence of both impaired
platelet production and increased platelet clearance. J Clin Invest
80:33, 1987
47. Karpatkin S, Strick M, Siskind GW: Detection of splenic
antiplatelet antibody synthesis in idiopathic autoimmune thrombocytopenic purpura (ATP). Br J Haematol23:167, 1972
48. Holme S, Heaton A, Kunchuba A, Hartman P: Increased
levels of platelet associated IgG in patients with thrombocytopenia
are not confined to any particular size class of platelets. Br J
Haematol68:431, 1988
49. Lynch DM, Howe S E Antigenic determinants in idiopathic
thrombocytopenic purpura. Br J Haematol63:301, 1986
50. Karpatkin S: Autoimmune thrombocytopenic purpura. Semin
Hematol22260,1985
51. Naidu S, Messmore H, Caserta V, Fine M: CNS lesions in
neonatal isoimmune thrombocytopenia. Arch Neurol40:552, 1988
52. Branehog I, Kutti J, Ridell B, Swolin B, Weinfeld A: The
relation of thrombokinetics to bone marrow megakaryocytes in
idiopathic thrombocytopenic purpura (ITP). Blood 45:551,1975
53. Garg SK, Lackner H, Karpatkin S: The increased percentage
of megathrombocytes in various clinical disorders. Ann Intern Med
77:361, 1972