Pediatr Blood Cancer 2006;47:710–713
Mechanisms of Action of Intravenous Immunoglobulin in the
Treatment of Immune Thrombocytopenia
Andrew R. Crow,1,2,4 Seng Song,2,4 Vinayakumar Siragam,1,2,4 and Alan H. Lazarus1,2,3,4*
Intravenous immunoglobulin (IVIG) is currently used to treat a
multitude of autoimmune disorders including immune thrombocy-
topenic purpura (ITP), yet the mechanism of action of IVIG remains
unresolved. Using a murine model of ITP in which IVIG functions
therapeutically, our laboratory has addressed such theories
as blockade/inhibition of the mononuclear phagocytic system,
cytokine regulation, and neutralization of pathogenic autoantibodies
mediated by anti-idiotypic antibodies, and these findings will be
Key words: immune complex; intravenous immunoglobulin; ITP
INTRODUCTION
Immune thrombocytopenic purpura (ITP) is an auto-
immune disease characterized by the production of platelet-
reactive autoantibodies which result in thrombocytopenia.
Platelet clearance occurs via phagocytosis in the mono-
nuclear phagocytic system (MPS). One successful treatment
for ITP and a host of other autoimmune diseases is intra-
venous immunoglobulin (IVIG). IVIG is prepared from large
pools of plasma, typically from more than 5,000 healthy
blood donors. The first description of the treatment of
individuals with ITP with IVIG was by Imbach et al. [1] who
reported that high-dose administration of IVIG resulted in a
rapid reversal of thrombocytopenia in children. IVIG’s exact
mechanism of action in ameliorating ITP remains poorly
understood. This review will address current research being
done on IVIG in ITP, with an emphasis on work being done in
our lab to understand IVIG’s mechanism of action and to
develop alternative therapies.
Mononuclear Phagocytic System (MPS) Blockade
It was initially postulated that the therapeutic activity of
IVIG in the amelioration of ITP was due to competitive
inhibition of activating Fcg receptors (FcgR) on phagocytic
macrophages within the MPS by IVIG-sensitized erythro-
cytes [2,3]. The most direct early evidence that MPS
blockade by IVIG could rescue antibody-sensitized cells
from phagocytosis was experiments carried out by Fehr et al.
[4], who showed that in patients with ITP who had not
undergone splenectomy, IVIG treatment prolonged the in
vivo clearance of radiolabeled, antibody-sensitized RBC. We
have also demonstrated that IVIG [5] and therapeutic soluble
immune complexes (sICs) [6] block the clearance of
opsonized, labeled RBC in a murine model of ITP.
Four classes of activating FcgR are expressed on
phagocytic effector cells in the MPS: the high affinity FcgRI,
which binds monomeric IgG, and the lower-affinity receptors
FcgRIIA, FcgRIII, and FcgRIV which bind complexed IgG,
ß 2006 Wiley-Liss, Inc.
DOI 10.1002/pbc.20980
discussed herein. We have also demonstrated that soluble immune
complexes can completely recapitulate the therapeutic effects of
IVIG in ITP, and recent work from us has identified activating Fcg
receptors on CD11cþ dendritic cells as the relevant molecular target
of IVIG in the acute resolution of murine immune thrombocytopenia.
This and other work to devise antibody-based IVIG alternative
therapies will also be addressed. Pediatr Blood Cancer
2006;47:710–713. ß 2006 Wiley-Liss, Inc.
such as immune complexes or IgG-opsonized platelets
(reviewed in [7,8]). Blocking the FcgRI seems to have no
effect in ITP [9] and IVIG ameliorates immune thrombocy-
topenia in mice genetically deficient in FcgRI [10]. Murine
studies have shown that administration of a blocking FcgRII/
III monoclonal antibody (2.4G2) prevented clearance of
IgG-sensitized RBC [11]. We have also shown that antibody
2.4G2 ameliorates murine immune thrombocytopenia,
although not nearly as efficiently as IVIG or other mono-
clonal antibodies which mimic the effects of IVIG or
polyclonal anti-D in the treatment of murine ITP [12,13].
Although competitive MPS blockade appears to be the
one of the most accepted mechanisms to explain the effects of
IVIG in ITP, some patients have responded to F(ab0 )2
fragments of IVIG, which have essentially no FcgR-binding
activity [14,15]. This suggests that other mechanisms may
contribute to inhibiting thrombocytopenia. In addition, a
recent report [16] has demonstrated that IVIG can ameliorate
thrombocytopenia induced by anti-platelet antibodies that
appear to function independent of FcgR expression [17]. It
has also been shown that IVIG is ineffective at treating ITP in
mice that lack the inhibitory receptor FcgRIIB [10]. We have
found that IVIG blocks the ability of the MPS to clear
labeled, opsonized RBC in these FcgRIIB deficient mice
(Fig. 1). Since mice that lack FcgRIIB do not benefit from
IVIG administration (discussed in the next section) but
nevertheless undergo MPS blockade by IVIG, either IVIG
— —————
1
The Canadian Blood Services, Toronto, Ontario, Canada;
2
Department of Laboratory Medicine, Toronto, Ontario, Canada;
3
Departments of St. Michael’s Hospital, Medicine, and Laboratory
Medicine and Pathobiology, University of Toronto, Toronto, Ontario,
Canada; 4The Toronto Platelet Immunobiology Group, Toronto,
Ontario, Canada
*Correspondence to: Alan H. Lazarus, Transfusion Medicine
Research, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario,
Canada M5B 1W8. E-mail: lazarusa@smh.toronto.on.ca
Received 20 June 2006; Accepted 20 June 2006

Fig. 1. IVIG blocks the MPS in FcgRIIB deficient mice. FcgRIIB
deficient (FcgRIIB
/
) mice were injected intraperitoneally with
nothing (Nil) or 2 g/kg IVIG (‘‘Gamimune’’ 5%, Bayer, Inc, Elkhart,
IN). Twenty-four hours later, all mice were injected intravenously
with RBC that had been opsonized with the RBC-specific monoclonal
antibody TER-119 and fluorescently labeled with the dye PKH26
essentially as described in [13]. At 3, 10, 30, 120, and 960 min
postinjection, all mice were bled and the total number of RBC as well as
the percentage of fluorescently labeled RBC were counted by flow
cytometry as described in [13]. The percentage of labeled cells at 3 min
was considered 100%. *P < 0.05, **P < 0.005 compared with Nil,
using Student’s t test. n ¼ 4 mice per time point.
does not function via MPS blockade, or RBC clearance, as
assessed by the MPS blockade assay, may occur by a different
mechanism than platelet clearance in murine ITP. Indeed,
preliminary experiments in our laboratory have found IVIG
ineffective at preventing RBC clearance in a murine model of
autoimmune hemolytic anemia (SS & AHL, unpublished
observations, 2006). Taken together, these data suggest that
either IVIG does not ameliorate thrombocytopenia by MPS
blockade or that antibody-mediated clearance of platelets
and RBC may occur via different pathways, and thus anti-D
and IVIG may indeed function by different, but not neces-
sarily mutually exclusive, pathways as we have recently
suggested [12]. These data suggest that while IVIG may
indeed block MPS function in normal mice, this event may
not be related to the platelet recovery seen in ITP.
Inhibitory FcgRIIB
The concept of MPS ‘‘blockade’’ has implied a more
passive role for IVIG, namely competitive inhibition by
IVIG-generated immune complexes and opsonized platelets
for occupancy of activating FcgRs in the MPS. However,
work from Samuelsson et al. [10] has shown that IVIG
requires the presence of the inhibitory IgG receptor,
FcgRIIB, for preventing the induction of thrombocytopenia
in a murine model of passive ITP. We have confirmed this,
and extended these observations by demonstrating that IVIG
also requires this receptor for elevating platelet counts in
mice with established immune thrombocytopenia [18]. How
IVIG functions via FcgRIIB is unclear. It is known, however,
that co-crosslinking of FcgRIIB with the B cell receptor
Pediatr Blood Cancer DOI 10.1002/pbc
Treatment of Immune Thrombocytopenia 711
complex or with FcERI in mast cells results in cell inhibition,
mediated by recruitment of the inositol phosphatase SHIP1 to
the cytoplasmic tail of the FcgR (reviewed in [19]). Whether
or not this pathway is involved in IVIG action, however, has
not been established, and we have found that the individual
expression of SHIP1, (and two other potential signaling
mediators, SHP-1 and Btk) are not required for IVIG action
in amelioration of immune thrombocytopenia [18], and thus
IVIG may use a more complicated mechanism to initiate the
amelioration of immune thrombocytopenia than solely
effects through the inhibitory FcgRIIB.
Other Mechanisms
IVIG has been demonstrated in a number of studies to
have immunological effects on the cellular immune response
itself (reviewed in [20]). While work in our laboratory has
revealed that neither B, nor T cells are required for the acute
protective effect of IVIG [5], and that IVIG acutely
ameliorates murine ITP independent of the presence of
TNF-a, IFN-g IL-12b, MIP-1a, IL-2, 4, 7, 9, 10, 15, or 21
[21], numerous studies have nevertheless demonstrated
modulation of synthesis and secretion of various pro- and
anti-inflammatory cytokines both in vivo and in vitro after
exposure to IVIG [22–24]. Whether or not these cytokines
are merely a side effect of IVIG administration or actually
play a role in IVIG function remains unclear.
Another proposed mechanism for IVIG function involves
the regulatory properties of a subset of antibodies called anti-
idiotypic antibodies, that is, antibodies which react with the
antigen combining (or idiotypic) region of other antibodies
(reviewed in [7,20]). The potential role that anti-idiotype
antibodies may play in the amelioration of ITP has been
difficult to ascertain with in vivo studies. Berchtold et al. [25]
demonstrated that IVIG contains antibodies that can bind to
and neutralize the effects of autoantibodies directed against
anti-aIIbb3 (glycoprotein IIb/IIIa, a major target of auto-
antibodies in ITP). In contrast to these findings, Barbano et al.
[26] found IVIG to be ineffective at autoantibody neutraliza-
tion in ITP patients. Work from our own laboratory has shown
that the anti-idiotype antibodies present in IVIG are not
required in the amelioration of thrombocytopenia a murine
model of ITP [5]. In addition, we found IVIG to be ineffective
at neutralizing the anti-platelet antibody used to induce
passive murine ITP, both in vitro and in vivo [5].
IVIG REPLACEMENT THERAPIES
Monoclonal IVIG
Polyclonal anti-D (a type of IVIG) consists of IgG
selectively taken from the plasma of donors immunized to the
D antigen. It was first used in ITP by Salama et al. [2,3] who
postulated that the success of IVIG in treating ITP was due to
competitive inhibition of the MPS by sensitized RBC. Since
anti-D is considered to be ineffective in patients who are Rh D
712 Crow et al.
antigen negative, MPS blockade has been considered to be
the major mechanism of action of anti-D. Work in our
laboratory has shown that IVIG can be replaced by
monoclonal anti-RBC (or ‘‘anti-D like’’) antibodies in the
treatment of murine ITP [13]. These antibodies were as
efficacious as IVIG in treating thrombocytopenia at dosages
of 1,000 times less than IVIG. These antibodies functioned
independent of FcgRIIB expression, and unlike IVIG,
significantly down-regulated the functional level of expres-
sion of the activating FcgRIIIA in splenic macrophages [12].
Thus not only do monoclonal anti-RBC antibodies offer a
potential substitute for IVIG or anti-D, but also due to the fact
that anti-D like antibodies appear to function by a different
mechanism than IVIG, they may be successful in patients that
are refractory to IVIG treatment.
Immune Complexes
IVIG can potentially bind to a number of different cell
surface or soluble antigens [27–29], and it has been reported
that these antibody specificities within IVIG may have
therapeutic effects through different mechanisms [30–33].
Recently our laboratory has demonstrated that antibodies
directed to a soluble antigen can reverse murine ITP [6]. In
these studies, we showed that mice treated with ovalbumin
(OVA)-specific IgG þ OVAwere protected from ITP, and that
these sICs required expression of FcgRIIB to function
therapeutically. Similarly, antibodies directed to murine
endogenous soluble antigens such as albumin and transferrin
also ameliorated ITP in an FcgRIIB-dependent manner.
These findings suggest that the therapeutic effects of sICs
mimic IVIG and, similar to IVIG, require the expression of
the inhibitory FcgRIIB. In addition, to extend our findings to
other autoimmune diseases, we have shown that anti-murine
albumin also protects against K/BxN mouse serum-induced
inflammatory arthritis, suggesting that IVIG activity in both
ITP and inflammatory arthritis may be associated with
the formation of small immune complexes [6]. Thus, IgG
antibodies directed to soluble antigens could potentially
augment or perhaps even replace current therapies such as
IVIG in the treatment of some autoimmune diseases.
Passive Transfer of IVIG-Primed Dendritic Cells
The exact cellular and molecular targets of IVIG in the
amelioration of ITP has remained, until recently, an enigma.
Recent work from our laboratory has shown that leukocytes,
incubated with IVIG or IVIG mimetics can, upon passive
transfer to naı̈ve mice, completely recapitulate the therapeu-
tic effects of IVIG in ameliorating thrombocytopenia [34].
Extending this finding further, we were able to determine that
activating FcgR on CD11cþ dendritic cells (DC) are the
relevant molecular target of IVIG in the rapid resolution of
murine ITP [34]. This finding suggests that IVIG may be
bypassed using DCs that have been pretreated with IVIG or
an IVIG mimetic (sICs, or a monoclonal antibody regime that
Pediatr Blood Cancer DOI 10.1002/pbc
engages and hyper-crosslinks activating FcgR), followed
by reinfusion of these DCs back into the individual. This
may provide a novel high-potency therapeutic for ITP and
possibly other autoimmune diseases as well.
In summary, while IVIG is used worldwide to treat ITP
and a multitude of other autoimmune diseases, its mechanism
of action remains unclear. IVIG demonstrates the ability
to block MPS function, regulate FcgR expression and to
modulate the immune system. Work by our group and others
has and will continue to shed light on the function of this very
complex therapeutic. Finally, although human-derived IVIG
is considered safe, there will always exist the possibility of
viral and/or prion contamination. Thus recombinant IVIG
replacement therapies such as monoclonal antibodies to
soluble antigens may not only prove to be more beneficial
therapies than IVIG but will, due to their recombinant nature,
will be free of any potentially harmful infecting contami-
nants.
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