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2006 107: 2976-2983
Prepublished online Nov 29, 2005;
doi:10.1182/blood-2005-06-2562
A novel murine model of fetal and neonatal alloimmune
thrombocytopenia: response to intravenous IgG therapy
Heyu Ni, Pingguo Chen, Christopher M. Spring, Ebrahim Sayeh, John W. Semple, Alan H. Lazarus,
Richard O. Hynes and John Freedman
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TRANSFUSION MEDICINE
A novel murine model of fetal and neonatal alloimmune thrombocytopenia:
response to intravenous IgG therapy
Heyu Ni, Pingguo Chen, Christopher M. Spring, Ebrahim Sayeh, John W. Semple, Alan H. Lazarus, Richard O. Hynes, and John Freedman
Fetal and neonatal alloimmune thrombocytopenia (FNAITP) is a life-threatening
bleeding disorder caused by maternal
antibodies directed against fetal platelet
antigens. The immunoreactive epitopes
in FNAITP are primarily located in the
extracellular regions of the platelet glycoprotein IIIa (!3 integrin). Here we have
established a novel animal model of
FNAITP using !3 integrin–deficient (!3"/")
mice. We demonstrated first that these
mice are immunoresponsive to !3 integrin; !3"/" mice transfused with wild-type
platelets generated specific anti–!3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, !3"/" female mice (both naive
and immunized) were bred with wild-type
male mice to recapitulate the features of
FNAITP. The titer of generated maternal
antibodies correlated with the severity of
FNAITP. High titer maternal anti–!3 anti-
bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even
miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and downregulated pathogenic antibodies in both
the maternal and fetal circulations. (Blood.
2006;107:2976-2983)
© 2006 by The American Society of Hematology
Introduction
Fetal and neonatal alloimmune thrombocytopenia (FNAITP) is an
alloimmune disorder which results from maternal antibodies that
cross the placenta, bind to fetal platelets, and mediate fetal platelet
destruction. The frequency of FNAITP is estimated at 0.5 to 1.5 per
1000 liveborn neonates.1,2 The major risk of FNAITP is intracranial
hemorrhage (ICH) with neurologic impairment or death. After
birth, ICH occurs in 10% to 20% of neonates with FNAITP, and
may be fatal in up to 5% of cases.3
There are at least 16 recognized human platelet antigens
(HPAs), and immunoreactivity to the different HPAs can cause
FNAITP.4 These antigens result from polymorphisms in the
glycoproteins (GPs) on the platelet surface such as GPIaIIa (!2"1
integrin), GPIb!, and GPIIbIIIa (!IIb"3 integrin). Amino acid
sequences inherited from the father that differ from those of the
mother may be targeted by the maternal immune system. Most
cases of FNAITP are due to incompatibility in the amino acid
sequence of the "3 integrin subunit. HPA-1a (polymorphism of
residue 33 in the "3 subunit) is the most common antigen causing
FNAITP in white newborns, accounting for 75% to 95% of clinical
FNAITP cases.5 HPA-4a (polymorphism in residue 143 of the "3
subunit) is the most common antigen causing FNAITP in Asian
newborns.6 In addition, incompatibility in residues 62, 140, 407,
489, 611, 633, and 636 of the "3 subunit has also been reported.4
Thus, a variety of alloantigens are located throughout the extracellular "3 integrin subunit and study of the immune response to the
entire "3 integrin subunit is of importance to the understanding
of FNAITP.
The process of the maternal immune response to fetal platelet
antigens is largely unknown. The mechanism by which alloantibodies cross the placenta is also not fully understood, although the
neonatal Fc receptor (FcRn) has been implicated as a receptor that
mediates placental immunoglobulin G (IgG) transport and controls
homeostasis of IgG levels in the circulation.7,8 Furthermore,
although it has been hypothesized that the mechanism of platelet
destruction may be similar to that of idiopathic thrombocytopenic
purpura (ITP),9 the pathogenesis of thrombocytopenia in FNAITP
has not yet been clearly established.
Effective therapy for FNAITP is currently limited. Compatible
(antigen-negative) platelets for transfusion are often difficult to
obtain on short notice. In contrast, intravenous IgG (IVIG) can be
readily and quickly made available. IVIG is thus an attractive
candidate for the treatment of FNAITP. While IVIG has been
reported to alleviate FNAITP, the results from different investigators are conflicting and no randomized trials have been reported.1,10
From the Departments of Laboratory Medicine and Pathobiology, Medicine,
and Pharmacology, University of Toronto, ON, Canada; The Canadian Blood
Services, Ottawa, ON, Canada; The Toronto Platelet Immunobiology Group, St
Michael’s Hospital, Toronto, ON, Canada; and the Department of Biology,
Howard Hughes Medical Institute and Center for Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA.
H.N. designed the experiments, analyzed data, and wrote the manuscript; P.C.
performed the research, analyzed data, and wrote the manuscript; C.M.S.
performed the research, and edited the manuscript; E.S. performed the
research, and analyzed data; J.W.S. analyzed data, and edited the manuscript;
A.H.L. analyzed data, and edited the manuscript; R.O.H. provided vital
reagents ("3#/# mice) and edited the manuscript; and J.F. provided analytic
tools (flow cytometer), analyzed data, and edited the manuscript.
Submitted June 29, 2005; accepted November 16, 2005. Prepublished online
as Blood First Edition Paper, November 29, 2005; DOI 10.1182/blood-2005-062562.
Supported in part by the start-up funds from Canadian Blood Services and St
Michael’s Hospital (H.N.); Dean’s Fund of University of Toronto (H.N.); Bayer/
Canadian Blood Services/Hema-Quebec/Canadian Institutes of Health
Research Partnership Fund (H.N., principal investigator [PI]; and J.F., co-PI);
and Canadian Institutes of Health Research grant no. 129403 (H.N., PI; and
J.F., co-PI); National Institutes of Health grant PO1-HL66105 (R.O.H.). E.S. is a
fellow of the Keenan Foundation at St Michael’s Hospital.
2976
Reprints: Heyu Ni, Canadian Blood Services and Dept of Laboratory Medicine
and Pathobiology, St Michael’s Hospital, University of Toronto, 30 Bond St Rm
2-006, Bond Wing, Toronto, ON, Canada M5B 1W8; e-mail: nih@smh.
toronto.on.ca.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
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BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
The mechanism of action of IVIG in the treatment of FNAITP and
ITP is under intensive study, but remains incompletely understood.11-13 Given the ethical difficulties in performing basic research on human fetuses and neonates with this life-threatening
disorder, an animal model of FNAITP would be very useful to
investigate the pathogenesis of the disorder and evaluate the
efficacy and mechanism of action of IVIG in FNAITP.
In this study, we established a novel murine model of FNAITP
that recapitulates features of the human pathologic condition, and
demonstrated that maternal IVIG administration has a systemic
effect on the amelioration of this disease.
Materials and methods
Mice
"3#/# mice were previously described14 and have been backcrossed onto a
BALB/c background; control wild-type (WT) BALB/c mice (6 to 8 weeks
of age) were purchased from Charles River Laboratories (Montreal, QC,
Canada). All mice were housed in the St Michael’s Hospital Research
Vivarium and the experimental procedures were approved by the Animal
Care Committee.
Reagents
IVIG and human albumin were obtained from Bayer Inc/Canadian Blood
Services (Elkhart, IN). Alkaline phosphatase–conjugated anti–goat and
anti–human IgG as well as anti–mouse polyvalent immunoglobulin and
FITC-conjugated anti–mouse IgG, were purchased from Sigma (St Louis,
MO). FITC-conjugated anti–mouse IgG1 and IgG2a as well as anti–human
IgG were purchased from BD Biosciences (Mississauga, ON, Canada).
Goat anti–human "3 integrin polyclonal antibody (sc-6627) and donkey
anti–goat IgG alkaline phosphatase were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Rat anti–mouse !IIb"3 integrin (JON2)
and GPIb! (p0p3) monoclonal antibodies were kindly provided by Dr
Nieswandt (Wurzburg, Germany). Bovine serum albumin (BSA), Tween20, and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) were purchased from Sigma.
Induction and treatment of neonatal alloimmune
thrombocytopenia
"3#/# female mice were immunized with WT mouse platelets in either 2 or
4 weekly transfusions (108 platelets/transfusion). After immunization, mice
were bled via the saphenous vein and sera were collected to test anti–mouse
"3 integrin IgG. The immunized female "3#/# mice were then bred with a
WT male mouse. After delivery, platelet counts and bleeding disorders in
pups, as well as IgG anti–mouse "3 integrin in both the mother and pups
were analyzed to determine whether the pups exhibited FNAITP. We also
set up breeding cages of WT $ WT, "3#/# $ "3#/# and naive "3#/# $ WT
mice as controls. For IVIG treatment, female "3#/# mice which demonstrated FNAITP in their first deliveries were injected intravenously with
300 %L of 10% IVIG (1 g/kg) each week after being bred with WT male
mice. Human albumin (1 g/kg) was used as a control. To confirm the effect
of IVIG, immunized "3#/# female mice were also treated with IVIG and
albumin after breeding with WT male mice during their first pregnancies.
Detection of anti–mouse !3 integrin antibodies in immunized
mice by flow cytometry
Blood samples were collected from saphenous veins of "3#/# mice which
were immunized with WT platelets. Sera were prepared by centrifuging
clotted whole blood at 13 400g for 5 minutes. IgG, IgG1, and IgG2a
antibodies were detected using a 1:100 dilution of sera which was allowed
to bind WT platelets at room temperature for 1 hour, centrifuged at 600g for
15 minutes, and washed with phosphate-buffered saline (PBS). The bound
antibodies were detected with FITC-conjugated goat anti–mouse IgG, rat
MURINE MODEL OF FNAITP
2977
anti–mouse IgG1, and IgG2a, respectively, and analyzed on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). The value for anti–platelet
IgG level was calculated as a fold increase ratio. Fold & mean fluorescent
intensity (MFI) of test serum/MFI of preimmune serum.
Immunoprecipitation
Designated primary antibody (4 %g of either a well-characterized monoclonal antibody to murine "3 integrin [JON2], or GPIb! [p0p3], or antisera
from the immunized "3#/# mice) and 20 %L of Protein G Sepharose beads
(Amersham Pharmacia Biotech, Baie d’Urfe, QC, Canada) were added to
lysates from 108 platelets and mixed end-over-end at 4°C for 1 hour. Beads
were pelleted at 2300g and washed 4 times with 800 %L of lysis buffer
(0.5% NP-40, 50 mM Tris, 150 mM NaCl, 1 mM PMSF, 5 %g/mL
leupeptin, and 1mM EDTA). Beads were then incubated in 60 %L of 2 $
protein sample buffer (0.004% bromophenol blue, 50 mM Tris [pH 6.8], 1%
SDS, and 5% sucrose) and boiled for 10 minutes. Beads were pelleted at
18 300g for 30 seconds, and the supernatant was loaded on a 7% denaturing
SDS–polyacrylamide gel electrophoresis (PAGE). Subsequently, the proteins were transferred and Western blotted in the same manner as described
in “Detection of anti–mouse "3 integrin antibodies by Western blotting.”
Detection of anti–mouse !3 integrin antibodies
by Western blotting
Mouse platelets (2 $ 106) were lysed, and the lysates from either WT or
"3#/# mice were separated on a 10% SDS-PAGE gel under nonreducing
conditions. After transfer to PVDF membrane (Hybond-P; Amersham
Pharmacia Biotech), membrane was immunoblotted with either antisera
from the immunized "3#/# female mice or sc-6627 goat anti–human "3
integrin antibody, which cross-reacts with murine "3 integrin, at room
temperature overnight, and incubated with alkaline phosphatase–conjugated anti–mouse polyvalent immunoglobulin or anti–goat IgG. Immunoreactive bands were developed by reaction with substrate (BCIP/NBT).
Induction of thrombocytopenia with antisera from immunized
!3"/" mice
WT BALB/c mice were bled via the saphenous vein and the initial platelet
count was determined for each mouse and 100 %L antisera or their dilutions
from immunized "3#/# mice (2- and 4-platelet transfusions) was injected
via the tail vein on day 1. Platelet counts for individual mice were
enumerated daily up to day 4.
Platelet enumeration
As previously described,11 whole blood (10 %L) was isolated from either
adult mice (saphenous bleeding) or pups (carotid bleeding) and diluted into
990 %L of 1% EDTA in PBS. The blood was then further diluted in PBS to a
final dilution of 1:12 000. A total of 20 to 30 %L of whole blood from the
pups was collected after carotid bleeding for platelet counts and antibody
detection. The samples were analyzed for 2 minutes on a flow rate–
calibrated FACScan flow cytometer, using forward scatter (FSC) versus
side scatter (SSC) to gate platelets. Reference samples were incubated with
FITC-conjugated anti–mouse CD61 antibody to identify the platelet
population.
Detection of free-circulating IgG against platelets and
platelet-associated IgG in heterozygous pups
To detect platelet-associated IgG (PAIgG), platelets were prepared from
5 %L whole blood from heterozygous pups and stained directly with
FITC-conjugated anti–mouse IgG for 30 minutes and analyzed with a
FACScan flow cytometer. For the determination of free-circulating antiplatelet IgG, sera from heterozygous pups were incubated with 106 WT platelets
at a 1:10 dilution for 1 hour, centrifuged at 600g for 15 minutes, and washed
with PBS. Binding of IgG anti–mouse platelet "3 integrin was assessed as
described in “Detection of anti–mouse "3 antibodies in immunized mice by
flow cytometry.”
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2978
NI et al
Detection of IVIG in maternal and neonatal circulations
by ELISA
Sera from heterozygous pups or female "3#/# mice were serially diluted in
PBS and coated (100 %L/well) on 96-well plates at 4°C overnight. Plates
were then washed 3 times with 0.5% Tween-20 in PBS and blocked with
2% BSA in PBS for 3 hours. Plates were then washed with 0.5%
Tween-20/PBS and incubated with 1:1000 alkaline phosphatase–conjugated goat anti–human IgG ('-chain specific) for 30 minutes. The color was
developed with p-nitrophenyl phosphate as substrate and the optical density
at 405 nm (OD405) values were recorded on a multiwell plate reader.
Anti-idiotype activity of IVIG to the anti–mouse !3
integrin antibody
IVIG (10 %g/mL) was preincubated with antisera from the immunized
"3#/# mice in test tubes at a ratio of 1:1 and incubated at 4°C overnight.
Samples were diluted to a final dilution of 1:100 and then added to WT
mouse platelets (106) for 1 hour at room temperature. Antisera and IVIG
alone were used as positive and negative controls, respectively. The
samples were centrifuged at 600g for 15 minutes, washed with PBS,
incubated with FITC-conjugated goat anti–mouse IgG or FITCconjugated monoclonal mouse anti–human IgG, and then assayed by a
FACScan flow cytometer.
Statistical analysis
Data are presented as means ( SEM. Differences between 2 groups were
assessed by Student unpaired t test or )2 test as indicated.
Results
!3"/" mice are immunoresponsive to platelet !3 integrin
Immune responsiveness of "3#/# mice to platelet "3 integrin
antigen is a prerequisite to establish a mouse model of FNAITP. To
test this immune responsiveness, we first immunized "3#/# mice by
BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
weekly transfusions of 108 WT platelets. The mice were highly
responsive to platelet "3 integrin antigen and antiplatelet antibody
was detected after 2 weekly transfusions of WT platelets. Both
IgG1 and IgG2a antibodies were detected (ie, both T-helper 1 [Th1]
and Th2-like immune responses existed in "3#/# mice; Figure 1A).
The antibodies were specific to "3 integrin since they did not
recognize either platelets from "3#/# mice or WT red blood cells
(Figure 1A). An increased titer of anti–"3 integrin IgG was found
after 4 weekly transfusions of WT platelets (Figure 1B), and these
antibodies were also detected by Western blot (Figure 1C).
Immunoprecipitating platelet lysates with either these antibodies,
the well-characterized rat monoclonal antibody JON2 (anti-mouse
"3 integrin), or p0p3 (anti-mouse GPIb! control), and subsequent
immunoblotting further confirmed the specificity of these antibodies (Figure 1D). Notably, the antibodies from the immunized mice
induced thrombocytopenia when injected into WT BALB/c mice
and a similar extent of thrombocytopenia was observed when
high-titer antisera from 4-times-transfused mice was diluted to a
titer comparable with that of the twice-transfused mice (Figure 1E).
Maternal anti–!3 integrin antibodies caused fetal and neonatal
bleeding disorders and promoted fetal miscarriage
To establish a murine model of FNAITP, and to determine if a
clinically relevant bleeding disorder could be observed during
pregnancy and following delivery, naive female "3#/# mice
(preimmunization) were bred with WT male mice. However,
anti–"3 integrin IgG was not detectable in these "3#/# mice after
the first and the second deliveries. Anti–"3 antibodies were
detected in the mothers at low levels by a flow cytometric assay and
Western blot in the third and subsequent deliveries (Figure 2A-B).
Correspondingly, platelet counts in pups from the first 2 deliveries
were not significantly decreased (Figure 3A), and bleeding disorders were not observed (Figure 3Bi). Since the female mice were
Figure 1. Murine anti–!3 integrin antibodies were generated after immunizing !3"/" female mice with wild-type mouse platelets. (A) WT mouse platelets were
incubated with a 1:100 dilution of sera from "3#/# mice immunized either 2 or 4 times weekly (dotted line indicates 2 times; bold line, 4 times) or preimmune sera (filled area) and
stained with FITC-conjugated goat anti–mouse IgG or rat anti–mouse IgG1 or IgG2a. "3#/# platelets or WT red blood cells (RBCs) were used as negative controls. (B) Titration of
IgG antibody in preimmune sera of "3#/# mice ("), and sera of mice after 2 (E) or 4 (#) WT platelet transfusions. (C) Western blot of platelet lysates with both control anti–"3
integrin antibody sc-6627 and antisera of mice after 4 WT platelet transfusions. No "3 integrin band was recognized by sera from nonimmunized "3#/# mice in the negative
control. (D) "3 integrin was immunoprecipitated from platelet lysates using either antisera from this study or the monoclonal antibody JON2 (anti-mouse "3 integrin) or p0p3
(anti-mouse GPIb!, negative control). The "3 integrin immunoreactive band was detected by both the antisera (left panel) and the positive control antibody sc-6627 (right panel)
in immunoblot analysis. (E) Thrombocytopenia was induced in WT BALB/c mice by 100 %L antisera and their dilutions from the "3#/# mice transfused 2 times (right panel) or 4
times (left panel) with WT platelets. PBS was used as a control. n & 3 in each group. Data are represented as means ( SEM.
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BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
Figure 2. Low-level antibody response in later pregnancies of naive female
!3"/" mice after breeding 3 times with wild-type male mice. (A) Detection of
antiplatelet IgG by flow cytometry. A 1:100 dilution of sera from the naive "3#/# mice
was incubated with WT mouse platelets. Anti–"3 integrin antibodies were detected at
very low levels in sera from the mice after 3 pregnancies (thin line) compared with
those from mice before breeding (filled area) by a flow cytometric assay. (B) Western
blot of platelet antigens with a 1:1000 dilution of sera from the naive "3#/# mice. "3
integrin was recognized by both the control antibody sc-6627 (data not shown) and
the antisera from the naive "3#/# mice after 3 pregnancies.
“old” and generated fewer pups after 3 to 4 deliveries, it was
difficult to compare the number of living pups and monitor their
bleeding disorders with controls. Therefore, we used immunized
female "3#/# mice in order to develop a mouse model for FNAITP
in which high-titer anti–"3 antibodies were generated and clinical
FNAITP indices were observed. This protocol may mimic those
women who have preconceptional exposure to "3 integrin via
either a previous pregnancy or exposure to sperm "3 antigen
(“Discussion”).
Figure 3. Spontaneous hemorrhage and thrombocytopenia in heterozygous progeny of immunized !3"/"
mothers. (A) Thrombocytopenia in heterozygous pups
delivered from immunized "3#/# mothers (transfused
twice with WT platelets) crossed with WT males. Pups
from breeding cages of WT $ WT, "3#/# $ "3#/#, and
naive female "3#/# $ WT were used as a control. Data
are presented as means ( SEM; n & 7-25 for each
group. (B) Spontaneous bleeding in heterozygous pups
delivered from immunized "3#/# mothers. (i) A heterozygous pup delivered from a naive "3#/# mother crossed
with a WT male as a healthy control. (ii) Bleeding in live
pups. (iii) Dead pups with massive ICH or abdominal
bleeding. Bleeding is indicated by arrows. (C) Massive
ICH and/or abdominal bleeding was found in fetuses in
"3#/# mice immunized with 4 weekly transfusions of WT
platelets. (i) ICH and abdominal bleeding. (ii) ICH.
MURINE MODEL OF FNAITP
2979
We set up 8 breeding cages (1 female and 1 male mouse per
cage) for WT males crossed with female "3#/# mice that had been
immunized twice with WT platelets. The titer of antiplatelet IgG in
these female mice was 1:800 (Figure 1B). We observed bleeding in
some of the delivered pups (Figure 3Bii-iii); abdominal and skin
bleeding as well as ICH were found in 7 of 54 live pups delivered
from 6 female "3#/# mice at the first delivery (Table 1). The
mortality rate was 24.1% (13 of 54 pups) due to internal organ
bleeding, and 2 female "3#/# mice had miscarriages (Table 1).
These findings were significantly different from those seen in the
naive group (preimmunization) in both mortality rate ()2 & 8.10,
P * .005) and incidence of bleeding ()2 & 7.89, P * .005).
Because the relationship between antibody titer and the severity
of FNAITP is controversial,15 we further studied "3#/# female mice
which were immunized 4 times with WT platelet transfusions. As
shown in Figure 1B, the titer of antibody in these mice was
approximately 4 times higher than that of the twice-immunized
mice. After breeding with WT male mice, 1 of the 3 immunized
female "3#/# mice died during delivery after a 3-week pregnancy.
Immediate autopsy showed that 3 mature-sized and 3 smaller
fetuses were present in utero. Severe bleeding including ICH,
abdominal hemorrhage, or both, was found in the 3 mature-sized
fetuses (Figure 3C). The other 2 pregnant female "3#/# mice had
abortions or miscarriages after a 2- to 3-week pregnancy. No live
pups were found and cannibalized remains of the neonates were
present, which reflected a mortality rate that was significantly more
severe than that of the twice-immunized mice ()2 & 4.96, P * .05).
We further examined the reactivity of antiplatelet antibodies
(immediately after delivery) from the 4-time-immunized mice, and
found that it was 3- to 4-fold higher than that of twice-immunized
mice. Thus, the titer of maternal IgG against "3 integrin correlated
with the severity of symptoms in this murine model (ie, 4-timeimmunized mice + twice-immunized mice + naive mice). Since
no living pups were delivered from the "3#/# mice with high-titer
antibody, we used female "3#/# mice transfused twice with WT
platelets for most of the remaining experiments.
Maternal anti–!3 integrin antibodies caused fetal
and neonatal thrombocytopenia
To test whether thrombocytopenia indeed occurred in neonates and
contributed to the mentioned bleeding disorders in this murine
model, we examined platelet counts in live heterozygous pups
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2980
BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
NI et al
Table 1. Effect of maternal anti–!3 integrin IgG and IVIG on fetal and neonatal bleeding disorders
Breeding
cages
Total
pups
Dead
pups
Bleeding
disorders
Miscarriage*
First delivery
"3#/# $ WT†
7
57
2#
0**
0
Immunized "3#/# $ WT‡
8
54
13#††
7**
2
Immunized "3#/# $ WT; IVIG§
2
12
0
0
0
Second delivery
Immunized "3#/# $ WT; IVIG$
5
36
1††
2‡‡
0
Immunized "3#/# $ WT albumin¶
2
16
1
4‡‡
0
Underlined value indicates that statistically significant difference was also observed compared with the immunized but IVIG-untreated group in the first delivery ()2 + 4.13;
P * .05).
*Female "3#/# mice delivered immature dead pups.
†Naive female "3#/# mice (without immunization with WT platelets) were bred with WT male mice.
‡Female "3#/# mice; first delivery after two times immunization with WT platelets.
§Female "3#/# mice were transfused with IVIG during first delivery.
$Female "3#/# mice were transfused with IVIG during second delivery.
¶Female "3#/# mice were transfused with albumin as a control during second delivery.
#P * .005. This and the following three footnotes indicate P values for comparisons of the two values in the table sharing that footnote.
**P * .005.
††P * .05.
‡‡P * .05.
delivered from female "3#/# mice immunized twice with WT
platelet transfusions. Platelet counts in the pups were significantly
decreased (132.2 ( 10.5 $ 109/L versus 618.3 ( 42.5 $ 109/L in
control mice; P * .001) (Figure 3A). We were not able to examine
the platelet counts in the dead pups, although more severe
thrombocytopenia may have been expected. The platelet counts of
pups delivered from naive female "3#/# $ WT male were similar
to those of the pups delivered from WT $ WT and "3#/# $ "3#/#
mice (Figure 3A). Thus, maternal antibodies of the pregnant "3#/#
mice, and not the genotype difference, were responsible for the
reduction of platelet numbers in the heterozygous pups.
Maternal anti–!3 integrin antibodies crossed the placenta
and bound fetal and neonatal platelets
Maternal antiplatelet IgG is the cause of FNAITP in human
patients. To test whether neonatal thrombocytopenia was induced
by maternal anti–"3 integrin antibody in our mouse model, we
examined circulating anti–"3 integrin IgG and PAIgG in heterozygous pups delivered from the immunized "3#/# mice. As expected,
antiplatelet IgG (Figure 4A) and IgG2a (MFI & 5.13 ( 0.2 vs
3.77 ( 0.1 in controls; P * .005) were detected in the sera of live
heterozygous pups. The level of antibody from pups was approximately 10% of maternal antibody, which is similar to the relative
antibody levels reported in human cases.16 Increased PAIgG was
found on the surface of platelets from live pups with either low
platelet counts and/or bleeding disorders (Figure 4B). There was no
antiplatelet antibody detected in the different control groups,
including pups delivered from the first 2 naive "3#/# female $ WT
male litters. These results confirm that, in our murine model,
maternal IgG crossed the placenta and bound to fetal platelets,
concomitant with the clinical manifestations of FNAITP.
demonstrate any bleeding disorders, 2 pups had minor bleeding
symptoms, and 1 pup was stillborn. The mortality rate was
significantly decreased compared with the first litters that did not
receive IVIG treatment (P * .05). The platelet counts of these pups
were elevated from 132.2 ( 10.5 $ 109/L to 353.3 ( 18.0 $ 109/L
(P * .001; Figure 5A). Notably, when albumin was used in place
of IVIG treatment in the same setting (second delivery), neonatal
platelet counts remained low (Figure 5A) and bleeding disorders
were similar to those of the first litters (Table 1). To exclude the
contribution of differing number of pregnancies in the IVIGmediated amelioration, 2 female "3#/# mice immunized twice with
WT platelets were injected intravenously with IVIG once a week
during their first pregnancies. The platelet counts of the pups
delivered from these female mice were also elevated to
IVIG ameliorated platelet counts and bleeding disorders
in the FNAITP murine model
We then evaluated the therapeutic efficacy of IVIG in our murine
model of FNAITP using "3#/# female mice that were transfused
twice with WT platelets. These mice (which previously delivered
FNAITP-affected neonates in the first pregnancy) were injected
intravenously with IVIG once a week after being bred with WT
male mice. As shown in Table 1, among the 36 pups which were
delivered from 5 IVIG-treated female mice, 33 pups did not
Figure 4. Circulating IgG and platelet-associated IgG in heterozygous pups
delivered from immunized !3"/" mothers. (A) Circulating IgG against mouse "3
integrin was detected in heterozygous pups (thin line) and pups of WT controls (filled
area) by flow cytometry. (B) Platelet-associated IgG was detected in heterozygous
pups (thin line) and pups of WT controls (filled area) by flow cytometry. Bar graphs
represent means ( SEM fold increase of circulating IgG and PAIgG from heterozygous pups versus WT pups; n & 5-12.
From www.bloodjournal.org at MASSACHUSETTS INST TECH on August 26, 2008. For personal use only.
BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
MURINE MODEL OF FNAITP
2981
in immunized "3#/# mothers during their first pregnancies (P * .05).
In control groups, which were treated with albumin, the level of
maternal IgG anti–"3 integrin did not significantly decrease in the
circulation (Figure 5B). We also found that IVIG administered to
pregnant mothers was able to cross the placenta since it was
detected in the sera of the heterozygous pups (Figure 5C).
To determine whether anti-idiotypic antibodies were present in
IVIG and played a role in decreasing the titers and attenuating
immunoreactivity of maternal IgG, sera from "3#/# mothers
immunized 4 times were preincubated with IVIG before being
incubated with WT platelets. No decrease of MFI was observed for
maternal IgG binding to platelets in the presence of IVIG. There
was also no IVIG binding to other portions (ie, agretope) of anti–"3
antibodies as determined by our flow-cytometric assay (Figure 5D).
We also did not detect IVIG binding to the antibodies generated
from "3#/# mothers that were immunized twice (data not shown).
These results suggested that anti-idiotypic antibodies were not
present in IVIG and may not be responsible for ameliorating
FNAITP in this murine model.
Figure 5. Effect of IVIG on neonatal platelet counts and maternal IgG levels. (A)
Platelet counts in pups from breeding cages of WT $ WT and "3#/# $ "3#/# mice as
a normal control, immunized female "3#/# $ WT mice (first delivery), immunized
female "3#/# $ WT mice (treated with IVIG or albumin in their first delivery), and
immunized female "3#/# $ WT mice (treated with IVIG or albumin in their second
delivery). n & 12-22. (B) IVIG decreased anti–"3 integrin IgG in the maternal
circulation. Sera of female "3#/# mice were incubated with 106 WT platelets at a final
dilution of 1:100 for 1 hour. IgG anti–mouse "3 integrin was detected by a flow
cytometric assay (n & 2-3). There was no significant difference in antibody level
between the first delivery (untreated) and the second delivery (albumin-treated), and
between the first delivery and second delivery after IVIG treatment. (C) IVIG was
detected by enzyme-linked immunosorbent assay (ELISA) in the sera of pups
delivered from the IVIG-treated mothers during their second pregnancy. Sera of pups
from the first delivery (ie, before IVIG treatment) were used as a negative control.
n & 3-5 mice. (D) Anti-idiotype activity of IVIG was not found. Preincubated sera from
immunized pregnant "3#/# mice with IVIG (dashed line; MFI & 145.2 in 1:100
dilution) did not decrease antibody-platelet binding activity compared with sera alone
(bold line; MFI & 152.0 in a 1:100 dilution). The thin solid line indicates IVIG alone
incubated with mouse platelets. The filled area indicates that anti–human IgG-FITC
did not bind to WT platelets incubated with IVIG plus antisera. Data are represented
as means ( SEM.
292.5 ( 23.3 $ 109/L (P * .001; Figure 5A) and bleeding disorders were attenuated (Table 1). Not surprisingly, bleeding disorders
and no significant decrease in circulating antibody (data not shown)
and no amelioration in platelet counts (Figure 5A) have been found
in the control albumin group. Furthermore, we tested the effect of
IVIG on 1 of the female "3#/# mice transfused 4 times with WT
platelets that had miscarriages during its first pregnancy. The
female mouse delivered 5 live pups without obvious bleeding
disorders in its second delivery, although the platelet counts of
these live pups were still low (198.6 ( 24.9 $ 109/L). These
results indicate that, in our mouse model, IVIG is able to: (1)
ameliorate the reduction of platelet counts; (2) ameliorate FNAITP
bleeding symptoms; and (3) reduce mortality and miscarriage.
IVIG decreased pathogenic antibodies in both the maternal
and neonatal circulations
As shown in Figure 5B, weekly administration of IVIG significantly decreased the level of IgG anti–"3 integrin in the immunized
"3#/# mothers during their second pregnancies (P * .005). The
level of anti–"3 integrin antibody (MFI) was decreased from 28.3to 4.6-fold relative to sera from the maternal circulation during
their first pregnancies. Furthermore, neither circulating antibody
nor PAIgG were detectable in the neonates (data not shown). A
similar down-regulatory effect of IVIG on antibody levels was seen
Discussion
In the present study, we report the first animal model of FNAITP.
Our results showed that murine antibodies against murine "3
integrin were generated in "3#/# mice. Breeding immunized female
"3#/# mice with WT male mice reproduced the clinically relevant
fetal bleeding disorders exhibited in human cases of FNAITP. This
model demonstrates: (1) maternal antiplatelet antibodies correlate
with the severity of the observed FNAITP phenotype; (2) maternal
IVIG administration has multiple effects on the amelioration of this
disorder, including decreased maternal antiplatelet antibody, depleted pathogenic antibody in the neonatal circulation, decreased
fetal platelet clearance, reduced bleeding disorders, and increased
fetal survival; and (3) the mechanism of action of IVIG is likely not
due to an anti-idiotype antibody effect. To our knowledge, this is
the first report that IVIG is able to decrease maternal and fetal
pathogenic antibody levels during pregnancy.
In human patients, approximately 50% of FNAITP cases occur
in the first pregnancy, and are apparent following delivery. It is
therefore usually difficult to identify these FNAITP patients and to
monitor their maternal immune responses during pregnancy.
FNAITP is often complicated with severe bleeding disorders,
including ICH and other internal organ hemorrhages. After diagnosis, immediate therapeutic action is required and it is ethically
impossible to set an untreated control to investigate this lifethreatening disease in the human population. Thus, an animal
model is important to study the pathogenesis of FNAITP and to
monitor potential therapeutic effects. While several models of other
immune thrombocytopenia have been reported,17-19 no previous
animal model of FNAITP exists.
We demonstrated that "3#/# mice were immunoresponsive
against murine "3 integrin and both Th1- and Th2-associated IgG2a
and IgG1 isotypes, respectively, were produced. Th1-like immune
responses have been shown to affect the pathogenesis of ITP and its
treatment.20,21 Although the relevance of both isotypes being
produced in FNAITP is unknown, the presence of complementfixing IgG2a may be of significance to the pathogenesis of FNAITP
in that platelet destruction can occur by at least 2 different
mechanisms (Fc-dependent phagocytosis and complement
activation).
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2982
BLOOD, 1 APRIL 2006 ! VOLUME 107, NUMBER 7
NI et al
After demonstrating immune responsiveness to "3 integrin, we
bred female naive "3#/# mice with WT male mice. In contrast to
human FNAITP, we did not observe significant bleeding disorders
following the first delivery, or even the second delivery. This
difference between mice and humans may be due to the short
period of pregnancy in mice; the time could be too short to generate
a significant immune response, in particular during the physiologic
immunosuppressive state of pregnancy.22 Another potential explanation is that many women may have been exposed to HPAs (eg, "3
integrin) prior to pregnancy. It has been reported that "3 integrin is
expressed in sperm.23 Thus, it is possible that preconceptional
intercourse may prime the human female immune system and
subsequent exposure to fetal "3 integrin alloantigen during pregnancy may boost the immune response and result in an FNAITP
phenotype. Our preliminary data that sera from some pregnant
women with FNAITP indeed recognized a sperm antigen at the
same molecular mass as "3 integrin (C.M.S. and H.N., unpublished
data, October 2004) support this hypothesis. The "3#/# female
mice immunized with WT platelet transfusions in this model may
mimic such preconceptional exposure (or that of human mothers
who have had previous pregnancies) in human FNAITP.
Our murine model was established by breeding "3#/# female
mice with WT male mice. This situation, however, may differ from
that of human patients. In human FNAITP cases, alloantibodies are
mainly formed against the polymorphic structure of the "3 integrin
chain, which might recognize different epitopes and therefore
might have different properties compared with the antibodies
developed in this animal model. In addition, although FcRn has
been reported to be responsible for maternofetal IgG transfer in
both human and rodents, transfer of IgG in humans shows
increased specificity (with respect to murine transfer), and there is
preferential transport of some isotypes over others.24 However, the
process of immune responsiveness and the pathogenesis of FNAITP
between human and mouse may be comparable. In fact, all clinical
indices and symptoms were well reproduced in this model. We also
found that maternal antibody titer correlated with the severity of
bleeding disorders and that a high titer of anti–"3 integrin antibody
may even induce miscarriage. The heterogeneity of the experimental picture in heterozygous pups is similar to that seen in humans.
The sites of bleeding may depend on the site of trauma during
pregnancy and delivery (eg, relative nonprotection of abdominal
organs or the large size of the head). Also, we speculate that some
anti–"3 antibodies may cross-react with endothelial cells and cause
vessel injury, which may enhance the severity of FNAITP and
affect the sites of bleeding.
The management of FNAITP is a challenge; currently, the most
effective antenatal therapy is weekly in utero platelet transfusions
using irradiated maternal/antigen-negative platelets. In addition to
technical difficulties associated with this procedure, this invasive
procedure may also cause bleeding, fetal trauma, and spontaneous
abortion. However, IVIG has been used to treat ITP patients since
198125 and later to treat FNAITP.1,10 The proposed mechanisms of
action of IVIG in ITP include: (1) reticuloendothelial system (RES)
blockade;11 (2) anti-idiotypic antibody activity;26 (3) induction of
T- and B-cell tolerance;27-29 (4) inhibition of dendritic-cell function;30 and (5) inhibition of macrophage phagocytosis via IVIG/
Fc' RIIB interaction.12,31 Recently, the role of FcRn in homeostasis
of IgG has been highlighted.32 FcRn may protect IgG from
proteolysis during transcytosis in epithelial and endothelial cells.33,34
Thus, IVIG may saturate FcRn and consequently promote the
accelerated clearance of pathogenic IgG.35,36 In fact, it has been
reported that IVIG may enhance the clearance of pathogenic IgG
by this mechanism in both ITP37 and rheumatoid arthritis.7
However, the mechanism of action of IVIG in FNAITP remains to
be elucidated. Although significant amelioration of FNAITP was
observed in our model, it is unclear whether: (1) T- and B-cell
tolerance is induced by IVIG in the maternal immune system,
which decreases pathogenic IgG production; (2) enhancement of
antibody proteolysis indeed occurs in pregnant mothers after IVIG
administration; (3) IVIG saturates FcRn in the placenta and blocks
maternal antibody transplacental transportation; (4) RES blockade
also occurs in the fetus; (5) the improved prognosis of maternal—
compared with in utero—IVIG administration in a previous case
report38 was due to the synergistic actions of the described
mechanisms; and finally (6) whether variation of the efficacy of
IVIG treatment in human FNAITP results from different sources
and dosages of IVIG39 used in different therapeutic regimens.
Our murine model offers a convenient means of addressing
these questions.
In summary, we have established an animal model of FNAITP
that reproduced the symptoms of human FNAITP. We demonstrated that anti–"3 integrin antibody may cause abortion and
miscarriage, and that maternal IVIG administration has a systemic
effect on amelioration of this disease. Our data that IVIG can
down-regulate pathogenic antibody in the maternal circulation may
have broad implications for other mother–antifetal antigen–related
diseases such as hemolytic disease caused by Rh antigen. This
model will be important for further investigation of the maternal
immune response and pathogenesis of FNAITP.
Acknowledgments
The authors would like to thank Dr Victor S. Blanchette and Dr
Gregory A. Denomme for their advice during the experiments. Dr
Bernhard Nieswandt provided anti–mouse GPIb! (p0p3) and
anti–mouse !IIb"3 integrin (JON2) monoclonal antibodies.
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