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RIIA in the Immune Clearance of Platelets: A Transgenic Mouse Model1
*
Department of Pediatrics, Jefferson Medical College, Philadelphia, PA 19107, and Hematology/Oncology Research, Alfred I. duPont Hospital for Children, Wilmington, DE 19899; and
Division of Hematology/Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIA, is expressed
on macrophages and platelets and may play an important role in the
pathophysiology of immune-mediated thrombocytopenia. Mice lack the
genetic equivalent of human FcRIIA. To better understand the role of
FcRIIA in vivo, FcRIIA transgenic mice were generated and
characterized. One transgenic mouse line expressed FcRIIA on
platelets and macrophages at levels equivalent to human cells, and
cross-linking FcRIIA on these platelets induced platelet
aggregation. Immune-mediated thrombocytopenia in this transgenic line
was studied using i.v. and i.p. administration of anti-mouse
platelet Ab. In comparison with matched wild-type littermates that are
negative for the FcRIIA transgene, Ab-mediated thrombocytopenia was
significantly more severe in the FcRIIA transgenic mice. In
contrast, FcR -chain knockout mice that lack functional expression
of the Fc receptors FcRI and FcRIII on splenic macrophages did
not demonstrate Ab-mediated thrombocytopenia. We generated FcRIIA
transgenic x FcR -chain knockout mice to examine the role of
FcRIIA in immune clearance in the absence of functional FcRI and
FcRIII. In FcRIIA transgenic x FcR -chain knockout mice,
severe immune thrombocytopenia mediated by FcRIIA was observed.
These results demonstrate that FcRIIA does not require the FcR
-chain for expression or function in vivo. Furthermore, taken
together, the data suggest that the human Fc receptor FcRIIA plays a
significant role in the immune clearance of platelets in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Therapy for immune thrombocytopenia (and to some extent other autoimmune disorders) is often directed toward reducing the Ab-mediated clearance by i.v. Ig, glucocorticoids, splenectomy, or other means (2). These seemingly distinct therapies work in large part by inhibiting the splenic macrophage Fc receptor-dependent clearance of these Ab-coated cells.
It has been well established that the Fc receptors for IgG (Fc
receptors) play a major role in immune clearance, in the accelerated
clearance of Ab-coated platelets, and in the therapeutic response
(10, 11, 12, 13, 14, 15, 16, 17, 18). For example, glucocorticoids diminish thrombocytopenia long
before they reduce IgG Ab titers. In fact, glucocorticoids have been
demonstrated to down-regulate macrophage and platelet surface Fc
receptor expression. Intravenous Ig and the administration of
anti-D Ig (coating Rh+ erythrocytes) also inhibit Fc
receptors, although additional mechanisms of action for i.v. Ig have
been postulated. Removal of the spleen and its Fc receptor-laden
macrophages is also of major therapeutic benefit in the treatment of
immune thrombocytopenia (19). A second line of evidence for the
importance of Fc receptors in immune clearance comes from studies in
mice. Mice deficient in Fc receptors do not become thrombocytopenic
when treated with anti-platelet Abs (20, 21).
The complexity of the human Fc receptor repertoire only
recently began to be appreciated (22, 23, 24, 25, 26, 27, 28). Human macrophages express on
their surface FcRI, FcRIIA, and FcRIIIA. Each is an activating
receptor that individually is capable of mediating phagocytosis
(29, 30, 31, 32, 33). FcRI and FcRIIIA require a FcR subunit, or -chain,
for expression and/or for signaling for phagocytosis in vivo (20, 34).
In contrast, FcRIIA does not require the -chain for its
expression and phagocytic activity, possessing IgG-binding and signal
transduction capabilities in the same molecule (30, 31, 32, 35). It is of
note that human platelets express FcRIIA as their sole Fc receptor
(29, 36, 37, 38, 39). When cross-linked, FcRIIA fully activates platelets
for secretion and aggregation (22, 23, 24, 40). The relative contributions
of the different Fc receptors to the pathophysiology and therapeutic
response in immune thrombocytopenia have not yet been dissected in
vivo.
Mouse models have been helpful in examining the pathophysiology of
immune clearance. FcR -chain knockout
(KO)3 mice have been
generated, and in these mice all activating Fc receptor functions
are abrogated in vivo (20, 34). In addition, there is no evidence of
immune thrombocytopenia after injection of anti-platelet Ab in
-chain KO mice (20). In contrast, matched heterozygous KO mice and
wild-type mice develop prominent Ab-mediated thrombocytopenia.
However, major differences exist between the Fc receptor repertoire
in mouse and humans, notably absence of FcRIIA on mouse macrophages
and absence of any mouse platelet Fc receptor (41, 42, 43, 44, 45, 46, 47). Therefore,
we sought to generate and characterize mice that express FcRIIA and
to test the hypothesis that expression of FcRIIA plays a role in the
pathophysiology of the thrombocytopenia caused by anti-platelet Ab.
In this work, we describe the generation of human FcRIIA transgenic
mice and examine the effects of FcRIIA expression on immune
clearance in normal mice and in -chain-deficient mice. The FcRIIA
transgenic mice more accurately reflect the human Fc receptor
repertoire and provide a model for evaluating the pathophysiology of
the immune clearance of platelets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIA transgenic mice
A P1 clone identified by PCR screening of a human P1 genomic
library was used to generate a 72-kb SfiI restriction
fragment that included the 20-kb FcRIIA gene, 45 kb of 5'-flanking
region containing the gene promoter that we have previously
characterized (48, 49), and 7 kb of 3'-flanking region (Fig. 1
A). The 20-kb gene includes eight exons: 1) 5'UT (UT,
untranslated); 2) 5'UT/S1 (S, signal peptide); 3) S2; 4) EC1 (EC,
extracellular domain); 5) EC2; 6) TM (TM, transmembrane domain); 7) C1
(C, cytoplasmic domain); and 8) C2/3'UT. The 72-kb DNA fragment was
used to create transgenic mice following established procedures at the
Transgenic Mouse Core Facility of The University of Pennsylvania
(Philadelphia, PA). All animals were studied under the guidance and
approval of the Institutional Animal Care and Use Committees at the
University of Pennsylvania and Alfred I. duPont Hospital for Children.
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RIIA gene by PCR
and Southern blot analyses, as follows. Genomic DNA was isolated from a
tail biopsy of 3-wk-old mice and amplified using a primer located in
the EC2 exon of the FcRIIA gene (PCR3) and another located in the
following intron (4INM). Primer sequences used for PCR and RT-PCR
experiments are shown in Table I. As an
internal control, a fragment of the mouse endogenous whey acidic
protein (WAP) gene was also amplified using a second set of primers,
WAP-S and WAP-A (50, 51). PCR was done for 30 cycles, with each cycle
consisting of denaturing at 94°C for 45 s, annealing at 56°C
for 30 s, and extension at 72°C for 1 min using a GeneAmp PCR
System 9600 (Perkin-Elmer, Norwalk, CT). PCR products were analyzed by
agarose gel electrophoresis for presence of the appropriately sized
FcRIIA band (280 bp). To verify that the transgene contained the
full-length FcRIIA sequence, additional primers were used to amplify
the 5'-untranslated region (T5S and 16 M) and the 3'-untranslated
region (4.0–7S and 4.0–6 M).
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RIIA gene as a template (24). The filter was washed in 2x
SSC/0.1% (w/v) SDS, 0.2x SSC/0.1% (w/v) SDS, and finally 0.1x
SSC/0.1% (w/v) SDS at 65°C, and then exposed to film at -70°C
with an intensifying screen. The intensity of bands on film was
analyzed by Imagequant PhosphorImager software (Molecular Dynamics,
Sunnyvale, CA). This calculation was verified by comparing transgene
intensity to known amounts of plasmid DNA on the same Southern blot.
Equivalent loading of DNA from genomic samples was verified by
hybridization to the labeled endogenous mouse ß-actin cDNA (50). Of
the four transgenic mouse lines, one (line F011) contained
10 copies, one (line F032) 4 copies, and the other two
(line F07 and line F040) 1 copy of the
transgene per haploid genome.
The FcR -chain KO mice were provided generously by Dr. Jeffrey
Ravetch (Rockefeller University, New York, NY). They were
analyzed by PCR of genomic DNA using primers specific for the wild-type
and KO gene loci. C57BL/6 x SJL F1 mice (B6SJL
F1) served as wild-type controls (The Jackson Laboratory,
Bar Harbor, ME).
The line 11 FcRIIA transgenic mice were crossed with the FcR
-chain KO mice and bred to be hemizygous for FcRIIA transgene and
homozygous for the -chain KO gene, as assessed by PCR of genomic DNA
and analysis of offspring.
For isolation of RNA and analysis of mRNA expression, whole blood (200
µl) was collected from each line of transgenic mice by puncture of
the retro-orbital sinus with heparinized hematocrit tubes (Fisher
Scientific, Pittsburgh, PA). Platelet-rich plasma was isolated by
centrifuging the blood samples at 100 x g in a Sorvall
RT6000B centrifuge (Sorvall, Newtown, CT). Platelets were then pelleted
at 1900 x g, and total RNA was isolated using RNA
STAT-60 (Tel-Test, Friendswood, TX) using the manufacturer’s protocol.
RT-PCR was then performed as follows: cDNA was made from total RNA
using random hexamers and M-MLV reverse transcriptase (Life
Technologies, Gaithersburg, MD). PCR was then used to amplify a section
of the FcRIIA cDNA using intron-spanning primers to distinguish cDNA
from genomic DNA signal. The sense primer chosen was located in the EC2
exon of the FcRIIA gene (PCR3), and the antisense primer was located
in the downstream TM exon (195 M). PCR was done for 35 cycles, with
each cycle consisting of denaturing at 94°C for 15 s, annealing
at 54°C for 30 s, and extension at 72°C for 45 s,
followed by a 72°C hold for 2 min using a GeneAmp PCR System 9600.
Products were analyzed by agarose gel electrophoresis for the presence
of the appropriately sized bands (300 bp). Primers to mouse ß-actin
mRNA (mßactinS and mßactinA) were chosen as controls for the
integrity of the mRNA through the RT-PCR process.
Transgene-encoded protein was detected on the surface of megakaryocytes
and macrophages by immunohistochemical staining of bone marrow
aspirates and spleen samples from transgenic mice. Tissues were
selected from B6SJL F1 wild-type mice as negative controls.
U937 cells, a monocyte-like cell line that expresses the FcRIIA
receptor, served as a positive control (29). Peritoneal macrophages
were induced with thioglycolate following standard procedures (52).
Macrophage surface receptor density was quantitated using Scatchard
analysis. 125I-IV.3 binding to transgenic mouse
macrophages, wild-type mouse macrophages, and human FcRIIA-positive
HEL cell line control was performed to measure FcRIIA receptor
density, as previously reported (53).
Bone marrow aspirates flushed from both femurs and humeri of transgenic or wild-type mice were placed in 1 ml PBS on wet ice. Unfixed samples were placed in a cytospin (Shandon Lipshaw, Pittsburgh, PA) at 150 µl per cyto-funnel and pelleted onto poly(L) slides at 1000 rpm for 10 min. Slides were immediately removed from the cytospin, wrapped in aluminum foil, and placed in a -70°C freezer. Suspended U937 cells were suspended 1:1 (v/v) with PBS. Approximately 100 µl was placed in each funnel, spun at 1000 rpm for 10 min onto poly(L) slides, and stored similarly to the bone marrow slides. Excised spleens from both transgenic and wild-type mice were snap frozen in isopentane chilled in liquid nitrogen, and stored at -70°C. Subsequently, 5-µm sections were cut (Reichert-Jung Frigocut, Leica, Deerfield, IL) and placed on poly(L) slides, which were also stored, wrapped in foil, and frozen at -70°C. Just before staining, all slides were air dried for 10 min and warmed to room temperature (RT).
All slides were briefly rinsed in PBS, then placed in Peroxo-Blocker
(Zymed, San Francisco, CA) for 1 min at RT. Slides were rinsed well in
PBS, and placed in CAS-Block (Zymed), avidin, and biotin blockers, each
for 10 min with several washes of PBS before and after each blocking
step. Slides were then placed in the primary Ab IV.3 (monoclonal
anti-human FcRIIA, mIgG2b), at a dilution 1/100 in 2% (v/v)
FBS. The sections were incubated (50–100 µl per section) in a humid
chamber at 37°C for 30 min, then at RT for an additional 30 min.
Negative controls that omitted the primary Ab were incubated similarly
in 2% (w/v) FBS and CAS-Block at 1:1. All slides were rinsed well with
PBS and placed in the secondary goat anti-mouse (GAM) at 1:500 in
CAS-Block in a humid chamber. The samples were incubated at RT for
1 h, rinsed well in PBS and deionized water, and detected with
streptavidin-AEC kit (Zymed). Slides were counterstained in Mayer’s
hematoxylin for 2 min at RT, rinsed in deionized water, and mounted in
Advantage aqueous mounting medium (Innovex, Richmond, CA). As a control
for identification of mouse macrophages, Ab to mouse FcRII/III
(2.4G2, rat IgG2b; PharMingen, San Diego, CA) was used as primary Ab.
FcRIIA protein expression and function on platelets
Transgene-encoded protein was detected on the surface of
platelets using several methods. In the immunoblotting technique, whole
blood was isolated from transgenic mice via cardiac puncture in 3.8%
(w/v) sodium citrate to prevent coagulation. Blood samples were spun at
100 x g in a Sorvall RT6000B centrifuge to isolate the
platelet-rich plasma. Platelet-rich plasma was centrifuged at 1900
x g to pellet platelets, and the platelet pellet was washed
once with PBS and again pelleted. Platelets were lysed using Triton
lysis buffer with protease inhibitors (1% (v/v) Triton X-100, 50 mM
Tris 7.6, 1 mM DTT, 10 µg/ml leupeptin, and 10 µg/ml aprotinin).
Following clarification by centrifugation at 15,000 rpm for 30 min at
4°C, cell lysates were resolved on a 7.5% (w/v) SDS-polyacrylamide
gel, electrophoretically transferred to nitrocellulose, and
immunoblotted with the anti-FcRIIA mAb II.1.A.5, provided by Dr.
Jurgen Frey (Universitat Bielefeld, Germany). Blots were developed with
horseradish peroxidase-conjugated GAM Ab (Bio-Rad, Richmond, CA).
Immunoreactivity was detected by enhanced chemiluminescence (ECL)
according to the manufacturer’s protocol (Amersham).
Platelet surface receptor density was quantitated using Scatchard
analysis. Platelets were prepared from whole blood, as indicated above,
counted, and resuspended in 1x Tyrode’s buffer. 125I-IV.3
binding to transgenic mouse platelets and human platelet controls was
performed to measure FcRIIA receptor density on the platelet
surface, as previously reported (53).
Function of the FcRIIA receptor on the transgenic mouse platelets
was tested in aggregation experiments using anti-human FcRII Ab
IV.3 (20 µg/ml) as the primary Ab (22, 23, 24, 25). A F(ab')2 GAM
Ab (40 µg/ml; Caltag, San Francisco, CA) was added as the secondary
Ab to promote cross-linking of the primary Ab. Whole blood was
collected via cardiac puncture from wild-type and transgenic mice in
3.8% (w/v) sodium citrate to prevent coagulation. The blood was warmed
for several minutes at 37°C and, following addition of primary and
secondary Ab or control aggregation reagents (1 U thrombin, 10 µM ADP
(final concentration), or 2 µg/ml collagen (final concentration)),
aggregation was measured in an aggregometer using measurement of the
impedance signal (ChronoLog, Havertown, PA), according to the
manufacturer’s protocol. Additional aggregation experiments were
performed in which 4A5 Ab was added (final concentration 20–40
µg/ml), and aggregation response was measured.
Injection of Abs into mice
Abs or controls were injected either i.p. or i.v., as follows. Mice from each line studied were given a single i.p. injection of 100 µl of PBS containing 70 µg of 4A5, a rat anti-mouse platelet Ab (42). 4A5 is well characterized, and the thrombocytopenia that results from 4A5 injection into wild-type mice has been reported previously by an independent group. The injection time is considered t = 0. The 4A5 Ab was purified from the supernatant of a culture of 4A5 hybridoma cells (generously provided by Dr. Sam Burstein of the University of Oklahoma) using a mAb Trap G II kit (Pharmacia, Piscataway, NJ), according to the manufacturer’s instructions. A control mouse from each line was injected with 100 µl of PBS containing no Ab. Another group of transgenic mice was injected with 100 µl of PBS containing 70 µg of a rat IgG2a isotype control Ab (PharMingen).
In separate experiments, transgenic mice and wild-type mice were given a single i.v. (tail vein) injection of 70 µg of 4A5 Ab in 50 µl of PBS. A control mouse from each line was given an i.v. injection with 50 µl of PBS containing no Ab.
Platelet counts
Platelet counts were obtained before and at timed intervals after injection of anti-platelet Abs. Whole blood (200 µl) was collected by puncture of the retro-orbital sinus of anesthetized mice using heparinized hematocrit tubes (Fisher Scientific). Platelet-rich plasma was isolated, and platelet counts were obtained using a Coulter Z1 counter set for a mouse platelet aperture (Coulter, Miami, FL). The platelet counts are reported in number/µl.
Statistical analysis
The nadir platelet count following anti-platelet Ab
injection was the basis for comparison. Platelet counts from groups of
6 to 12 FcRIIA transgenic, -chain KO, (FcRIIA transgenic
x -chain KO), and wild-type mice each were compared using ANOVA
analysis, with two-sided p < 0.05 considered
significantly different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIA transgenic mice
The FcRIIA gene encompasses 20 kb and includes eight exons (48)
(Fig. 1
A). A 72-kb P1 genomic
clone fragment was used for creation of transgenic mice. This genomic
clone encodes the R131 polymorphic form of human FcRIIA,
which binds the widest range of mouse IgG subclasses, specifically
binding mouse IgG1 and rat IgG2a (the 4A5 isotype) well (54). We
produced four FcRIIA transgenic mouse founders (B6SJL genetic
background) containing the entire FcRIIA gene, as assessed by
positive PCR reactions with three different sets of primers, from the
middle of the gene (Fig. 1B) as well as the most 5' and 3'
ends. Southern blot analysis confirmed the presence of the transgene,
the absence of any major gene rearrangement, and estimation of
transgene copy numbers ranging from 10 (F011) to 4
(F032) to 1 (F07 and 40) (data not shown). Each
FcRIIA transgenic mouse founder demonstrated germ-line transmission
of the transgene.
FcRIIA mRNA expression was studied in each of the four transgenic
mouse lines using RT-PCR. Platelets were isolated from whole blood, RNA
was prepared from the platelets, and RT-PCR with primers specific for
human FcRIIA was performed. All four transgenic mouse lines
expressed human FcRIIA mRNA in their platelets (data not shown).
Tissue and cellular distribution of the FcRIIA protein were studied
by immunohistochemistry using IV.3 Ab. Controls included nontransgenic
mice and studies in which the primary Ab was omitted. FcRIIA
expression was found in marrow megakaryocytes and leukocytes
(identified in other sections with 2.4G2 Ab to the endogenous mouse
FcRII/III) in our transgenic animals (Fig. 2a). FcRIIA expression in
megakaryocytes and macrophages was also detected in the spleen (Fig. 2c), while the lymphoid cells in the follicles showed no
expression. Colocalization of 2.4G2 staining (anti-murine
FcRII/III) and human FcRIIA (125I-IV.3 binding) was
shown on the same set of thioglycolate-induced peritoneal macrophages
from line 11 transgenic mice (Fig. 2, e and f).
The Scatchard analysis indicates 
65,000 IV.3 binding sites on
the surface of line 11 transgenic mouse macrophages, equivalent to the
receptor level on human macrophages (55).
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RIIA protein expression with several
assays. Mice from the transgenic lines were positive for platelet
membrane FcRIIA expression, as assessed by immunoprecipitation of
platelet lysates with the anti-human FcRIIA mAb IV.3, followed
by immunoblot with a second anti-FcRIIA Ab (II.1A.5).
Immunoprecipitates from the transgenic lines contained a protein whose
size was identical to the human FcRIIA receptor, 40 kDa (data not
shown). Platelet FcRIIA surface receptor density measured using
125I-IV.3 binding showed 1550 sites per platelet in the
hemizygous line 11, which is in the normal range for human platelets
(Fig. 3A).
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RIIA gene copy number, produced the
highest qualitative RT-PCR signal in RNA analysis, and had the highest
protein signal in whole platelet ELISA and
immunoprecipitation/immunoblot experiments. We also demonstrated by
Scatchard analysis that the FcRIIA protein level on line 11
platelets is within the normal range for human platelets. This line was
chosen as the major FcRIIA transgenic mouse line for further studies
in vitro and in vivo. Platelet aggregation studies
We examined platelet aggregation to determine function of the
FcRIIA receptor on platelets of transgenic mice. Human platelets,
but not wild-type mouse platelets, aggregated in response to treatment
with anti-human FcRIIA mAb IV.3, followed by cross-linking with
F(ab')2 GAM Ab. As a positive control, platelets from both
humans and wild-type mice aggregated in response to thrombin, ADP, or
collagen stimulation, as described in Materials and Methods.
Platelets from line 11 were reproducibly activated in the aggregation
assay with IV.3 + GAM cross-linking (Fig. 3B). Additional
aggregation experiments were performed in which 4A5 Ab was added (final
concentration 20–40 µg/ml), and the aggregation response was
measured. There was no aggregation of platelets from wild-type or
FcRIIA transgenic mice (Fig. 3B). This agrees with the
original description of the 4A5 Ab by another group, in which the Ab
did not cause platelet aggregation/activation of platelets of wild-type
mice (42). Addition of ADP following the absence of activation in
response to 4A5 confirmed the ability of those platelets to be
activated.
Immune clearance of platelets following injection of anti-platelet Abs
As a model for immune clearance, we determined the clearance of
platelets following injection of anti-platelet Abs. Wild-type and
transgenic mice were treated with anti-platelet Ab, 4A5, which is
known to induce moderate thrombocytopenia in wild-type mice (42).
Wild-type mice had moderate thrombocytopenia, reaching platelet counts
in the 600,000/µl range, as previously reported. In contrast,
FcRIIA transgenic line 11 mice exhibited significantly more severe
thrombocytopenia in vivo than wild-type mice following i.v. Ab
injection (p < 0.05, Fig. 4). Saline-injected controls showed no
significant change in platelet count.
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). The rebound rise
in platelet count is in agreement with earlier studies of Ab-mediated
thrombocytopenia (41). Intraperitoneal injections of anti-platelet
Ab showed a similar decrease in platelet counts, with platelet
clearance over days rather than over hours. As with i.v. injections,
i.p. injections of anti-platelet Ab caused immune thrombocytopenia
that was more profound in the FcRIIA transgenic animals
(p < 0.05, Fig. 5). Neither saline injection nor
injection of an isotype control Ab caused thrombocytopenia. Transgenic
line 11 mice demonstrated the most severe thrombocytopenia. The
thrombocytopenia in FcRIIA line 32 mice was also significantly more
severe than that in wild-type mice (p < 0.05),
indicating that the immune clearance of platelets mediated by FcRIIA
is evident in two independent transgenic lines. There was no
statistically significant difference in the nadir platelet counts
induced by 4A5 between FcRIIA transgenic line 11 and line 32.
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-chain KO mice, extending published observations with another
anti-platelet Ab and confirming that the mechanism of
thrombocytopenia is through Fc receptor-mediated clearance (20).
Immune clearance in FcRIIA transgenic x FcR -chain KO
mice
FcRIIA transgenic mice were bred to FcR -chain KO mice to
examine the role of FcRIIA in immune clearance in vivo in the
absence of FcRI and FcRIII. As described in Materials and
Methods, the mice tested were homozygous for the -chain KO
(-chain null or -/-) and hemizygous for the FcRIIA
transgene. In contrast to the -chain KO mice, these IIA tg
x -chain KO mice experienced severe thrombocytopenia when injected
with anti-platelet Ab. The nadir platelet counts averaged
73,000/µl (n = 6, range = 37,000–114,000/µl),
which is significantly different from -chain KO mice treated with
anti-platelet Ab (p < 0.05; Figs. 5 and 6). The nadir platelet counts were
comparable with that of the FcRIIA transgenic mice that coexpress
FcRI/ and III/. Since FcRI and FcRIIIA are deficient in
FcR -chain KO mice, these experiments provide evidence for a
critical role for FcRIIA in the immune clearance of platelets in
vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIA
transgenic mice. We generated a transgenic mouse line in which the
expression of the FcRIIA receptor in spleen macrophages and
platelets more closely recapitulates the expression in humans. These
mice allowed us to test the hypothesis that expression of FcRIIA
plays a role in immune clearance.
In our study, we examined thrombocytopenia due to anti-platelet Abs
as a model of immune thrombocytopenia. Although there are a number of
reported murine models of immune thrombocytopenia (20, 21, 41, 42, 43, 44, 45, 46),
none of them possesses the Ab effector mechanisms found in humans. We
have demonstrated that thrombocytopenia due to anti-platelet Ab is
more profound in the presence of FcRIIA expression. This was
observed for both i.v. and i.p. Ab administration in our primary
transgenic line. A second independent FcRIIA transgenic line also
demonstrated more severe thrombocytopenia due to anti-platelet Ab.
We found the same moderate thrombocytopenia induced by
anti-platelet Ab in wild-type mice as originally reported (42), and
observed no anti-platelet Ab-induced thrombocytopenia in FcR
-chain KO mice.
Our creation of the genetic cross of the FcRIIA transgenic mice with
the FcR -chain KO mice further provided the opportunity to study the
contribution of FcRIIA as an important Fc receptor in Ab-induced
thrombocytopenia. These genetically crossed mice developed immune
thrombocytopenia as profound as FcRIIA transgenic mice in which
murine FcRI and FcRIII were coexpressed. FcRI and FcRIII
are deficient in FcR -chain KO mice, while murine FcRIIB is
expressed. Murine FcRIIB is a regulatory Fc receptor that has an
inhibitory function (28, 32, 56). Thus, in the FcRIIA
transgenic x FcR -chain KO mice, FcRIIA is the major
activating Fc receptor. Our data provide the first evidence of the
significant role for FcRIIA in immune clearance in vivo and
independent of FcRI, FcRIII, and the FcR -chain.
Involvement of FcRIIA with the -chain has been proposed as a
result of experiments suggesting functional interaction in transfected
B cells (57), although FcRIIA does not require the -chain for
phagocytosis in vitro (30, 31, 32). Our studies in vivo clearly demonstrate
that the -chain is not necessary for the effect of FcRIIA in
immune clearance.
The FcRIIA transgenic mice represent an advance in mouse models of
human immune thrombocytopenia. Our data suggest that therapies to
diminish Ab effector mechanisms in human immune thrombocytopenia should
be directed at the expression or function of FcRIIA as well as the
other Fc receptors. Our FcRIIA transgenic mouse model provides a
mechanism for testing potential therapeutic modalities. Since these
mice express the activating platelet Fc receptor, they may also be
useful in studies of immune thrombocytopenias accompanied by
thrombosis, such as heparin-induced thrombocytopenia with thrombosis in
which the platelet Fc receptor is known to play a pathophysiologic role
(5, 6, 58). We cannot preclude any 4A5-induced platelet activation in
vivo, but that is unlikely given the result that 4A5 did not activate
platelets of wild-type or transgenic mice in vitro.
While we have demonstrated the importance of FcRIIA in vivo in the
immune clearance of platelets, the relative contribution of platelet
and/or macrophage FcRIIA expression to the pathophysiology of the
thrombocytopenia is uncertain. It is likely that molecular genetic
approaches will help dissect the comparative roles of these two
lineages. We recently identified sequences that mediate
megakaryocyte-specific expression in transgenic mice in vivo using the
PF4 gene promoter (50), as well as the promoter sequences responsible
for the regulation of FcRIIA transcription in vitro in
megakaryocytic and myelomonocytic cell lines (Cassel et al., manuscript
in preparation). Application of this knowledge to achieve selective
expression of FcRIIA in the platelet or spleen macrophage in vivo
should help address the role of these lineages in immune
thrombocytopenia.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven E. McKenzie, Hematology/Oncology Research, Alfred I. duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE 19899. E-mail address:
3 Abbreviations used in this paper: KO, knockout; GAM, goat anti-mouse; 125I-IV.3, 125I-labeled IV.3; RT, room temperature; tg, transgenic; WAP, whey acidic protein.
Received for publication October 2, 1998. Accepted for publication January 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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X-HaeIII DNA size markers.
