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RIII (CD16)1
*
Department of Immunology and
Medarex Europe, University Hospital Utrecht, Utrecht, The Netherlands;
Department of Clinical Immunology, Medical School Hannover, Hannover, Germany; and
§
Department of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIII
in mice, suggesting that IgG1 may interact preferentially with
FcRIII. In the present study, the biologic relevance of this
observation was addressed by triggering various effector functions of
the immune system in FcRIII-/- mice, using panels of
mAbs of different IgG subclasses. Both binding and phagocytosis of
IgG1-coated sheep or human erythrocytes by FcRIII-/-
macrophages in vitro were strongly impaired, indicating that the
impaired ingestion of complexed IgG1 by FcRIII-/-macrophages is due to a defect in binding. An in vivo
consequence of the defective phagocytosis was observed by resistance of
FcRIII-deficient mice to experimental autoimmune hemolytic anemia,
as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by
liver macrophages. Furthermore, trapping of soluble IgG1-containing
immune complexes by follicular dendritic cells in mesenteric lymph
nodes from FcRIII-/- mice was abolished. Whole blood
from FcRIII-/- mice was unable to induce lysis of
tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs
proved unable to mount a passive cutaneous anaphylaxis in
FcRIII-/- mice. Together, these results demonstrate
that IgG1 complexes, either in particulate or in soluble form, trigger
in vitro and in vivo immune effector functions in mice predominantly
via FcRIII. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R), which are expressed on leukocytes (1, 2). The interaction
between IgG and FcR, resulting in cross-linking of these receptors,
triggers various immune effector functions, such as the release of
toxic oxygen metabolites or inflammatory cytokines, degranulation,
phagocytosis, or Ab-dependent cellular cytotoxicity
(ADCC)3 (3, 4).
Three classes of FcR have been identified on murine leukocytes:
FcRI, FcRII, and FcRIII (3, 4). It has been proposed that both
murine IgG1 and IgG2b interact preferentially with the low affinity
receptors FcRII and FcRIII, and IgG2a with the high affinity
receptor FcRI (3). These patterns of interaction have been largely
based on in vitro studies using transfected cell lines. The precise
contribution of the interactions between each of the specific IgG
subclasses and the different FcR classes to in vivo immune reactions
thus remains to be clarified.
Recently, knockout mouse strains lacking individual FcR
classes have been generated, facilitating evaluation of IgG-mediated
immune effector functions under physiologic conditions. The knockout
strains generated are either deficient in the common FcR-signaling
subunit, the FcR -chain (lacking expression of Fc
RI, FcRI, and
FcRIII) (5), in FcRI (6), FcRII (7), or in FcRIII (8). Very
recently, the use of mice deficient in FcRI led to the finding that
murine IgG3 can selectively interact with this receptor (6).
Using mice lacking FcRIII, we have demonstrated that FcRIII is
the main FcR triggering passive cutaneous anaphylaxis and
complement-independent Arthus reaction, induced by polyclonal rabbit
IgG (8). In addition, we observed that in vitro phagocytosis of mouse
IgG1-, but not IgG2a- or IgG2b-coated sheep erythrocytes by exudate
peritoneal macrophages requires the presence of FcRIII. The latter
observation suggests an apparent specificity of complexed IgG1 for
FcRIII, and raises the question whether this finding merely
represents an in vitro phenomenon or whether it has broad implications
for the functioning of the immune system in vivo. In the present study,
we have extended this previous observation to various effector
functions of the immune system to assess its biologic relevance. Using
FcRIII-deficient mice and a variety of mAbs of different IgG
subclasses, we studied binding and ingestion of IgG-opsonized
erythrocytes by peritoneal macrophages in vitro, erythrophagocytosis by
liver macrophages in vivo, trapping of immune complexes by follicular
dendritic cells (FDC) in organ sections, lysis of tumor target cells by
whole blood, and in vivo passive cutaneous anaphylaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice deficient in FcRIII were generated as described (8).
Briefly, the ligand-binding EC2 domain and the transmembrane region of
the FcRIII gene were replaced by the hygromycin resistance gene by
homologous recombination in ES cells. Targeted cells bearing the
mutated FcRIII allele were injected into C57BL/6 blastocysts, and
chimeric mice were crossed with C57BL/6 mice; to establish a homozygous
mutant mouse strain, F2 heterozygous mice were
intercrossed.
Antibodies
Murine anti-human glycophorin A mAb, i.e., AME37-6298
(IgG1); AME37-6295 (IgG2a); and AME37-6480 (IgG2b) were derived from
Central Laboratory of The Netherlands Red Cross Blood Transfusion
Service (Amsterdam, The Netherlands). Murine anti-trinitrophenyl
(TNP) mAb used were H5 and D10 (IgG1), 7B4 and Hy1.2 (IgG2a), and 1B4
(IgG2b) (9), which were kindly provided by Dr. B. Heyman (Uppsala
University, Uppsala, Sweden). Murine anti-DNP mAb SPE-7
(IgE) was purchased from Sigma (St. Louis, MO). Murine mAb directed
against the extracellular part of anti-HER2/neu were TA1
(IgG1; derived from Oncogene Research Products, Cambridge, MA) (10),
520C9 (IgG1; derived from Medarex, Annandale, NJ) (11), and 13D1
(IgG2a; kindly provided by Dr. H. J. Bühring, University of
Tübingen, Tübingen, Germany) (12). Rat mAb 2.4G2 is
directed against murine FcRII and FcRIII (kindly provided by Dr.
J. Unkeless, Mount Sinai School of Medicine, New York,
NY) (13). Murine mAb directed against murine erythrocytes were
105-2H (IgG1) and 34-3C (IgG2a) (kindly provided by Dr. S. Izui,
University of Geneva, Geneva, Switzerland) (14).
Rosette formation between HRBC and macrophages
Human blood was washed three times with PBS, and HRBC were opsonized with mAb against human glycophorin A at subagglutinating concentrations for 30 min at 37°C, washed three times, and suspended at a concentration of 2% pelleted cells in RPMI with 10% FCS. To isolate peritoneal macrophages, mice were injected i.p. with 1 ml of 0.5% (w/v) thioglycolate (Difco Laboratories, Detroit, MI). Three or four days later, peritoneal macrophages were isolated, washed three times with ice-cold PBS by centrifugation for 5 min at 300 x g and 4°C, and suspended at 2 x 105 cells/ml of RPMI containing 10% FCS. To prevent ingestion, the cells were incubated with 2 µg/ml of cytochalasin D (Sigma) for 30 min at 37°C. Next, 100-µl suspensions of macrophages were mixed with 25 µl of opsonized HRBC in the presence of 2 µg/ml of cytochalasin D, sedimented by centrifugation for 4 min at 33 x g at room temperature, and incubated for 30 min at 37°C. The percentages of macrophages appearing in rosettes were determined by light microscopy.
Binding and phagocytosis of SRBC by adherent macrophages
SRBC were washed three times with PBS, and were conjugated with TNP by incubation in a solution of 3.6 mg trinitrobenzene sulfonic acid/ml of 0.28 M cacodylate buffer (pH 6.9) for 15 min at room temperature. Next, the SRBC were washed thrice with PBS and once with 1% glycyl glycin. TNP-conjugated SRBC (TNP-SRBC) were opsonized by incubation of 0.5% pelleted TNP-SRBC with 5 µg/ml of purified anti-TNP mAb or with 2x diluted hybridoma culture supernatant for 30 min at 37°C, followed by three washes. Thioglycolate-elicited macrophages, isolated as described above, were allowed to adhere by incubation of 105 cells in RPMI 1640 medium containing 10% FCS/well of a 96-well tissue culture plate (Nunc, Roskilde, Denmark) for 3 h at 37°C. Nonadherent cells were removed by two washes. To study binding of SRBC, the macrophages were incubated with 2 µg/ml cytochalasin D for 30 min at 37°C. Then, 50 µl of 1% pelleted opsonized SRBC-TNP, in the presence of 2 µg/ml cytochalasin D, was added to each well and incubated for 30 min at 37°C, and nonbound SRBC were removed by three washes with PBS. To study phagocytosis of SRBC, 50 µl of 1% pelleted SRBC in RPMI containing 10% FCS was added to each well and incubated for 30 min at 37°C, followed by lysis of extracellular SRBC by incubation in water for 1 min at room temperature and three washes with PBS. The percentages of macrophages that had bound or ingested one or more SRBC were determined by light microscopy.
In vivo erythrophagocytosis
Hemolytic anemia was induced by a single i.p. injection of pathogenic murine anti-murine erythrocyte mAb 105-2H (450 µg/mouse) or 34-3C (120 µg/mouse), as described (14). Mice were sacrificed 2 days later, and the livers were processed for histologic examination. Tissues were fixed in 10% buffered Formalin, embedded in paraffin, and stained with hematoxylin and eosin, according to conventional procedures.
Trapping of immune complexes
Mesenteric lymph nodes and spleens were excised and frozen in liquid nitrogen. Cryostat sections (8 µm) were cut and picked up on slides, which were fixed in acetone for 10 to 30 min, and air dried. Immune complex trapping was determined as described previously (15, 16), except that a mixture of trinitrophenylated peroxidase (TNP-PO, a generous gift of Dr. J. Laman, TNO Prevention and Health, Leiden, The Netherlands) and murine anti-TNP mAb was used. Briefly, the sections were overlayed with a solution containing 20 µg/ml of H5 (IgG1 anti-TNP), 7B4 (IgG2a anti-TNP), or 1B4 (IgG2b anti-TNP), and 10 µg/ml TNP-PO in 0.1% BSA in PBS (PBS-BSA) in the absence of serum to avoid involvement of complement activation, and incubated overnight at 4°C. Serial sections of one follicle were used for each of the IgG Abs. Next, the sections were rinsed extensively with PBS, and peroxidase activity was revealed by incubation with 0.5 mg/ml 3,3'-diaminobenzidine-tetra-HCl (Sigma) and 0.03% H2O2 in 50 µM Tris-HCl (pH 7.6). Control sections were incubated with or without TNP-PO in the absence of mAb. Sections were counterstained with hematoxylin. Immunohistochemistry was performed by an indirect immunoperoxidase method, as described (15, 16). Briefly, acetone-fixed sections were incubated with optimal concentrations of 2.4G2 in PBS-BSA for 1 h at room temperature, washed, and incubated with peroxidase-conjugated rabbit anti-rat IgG in PBS-BSA containing 5% normal mouse serum. Peroxidase activity was visualized as described above.
Lysis of IgG-coated tumor target cells by whole blood
Ab-mediated lysis of target cells by whole blood was determined using a standard short-term chromium release assay, as described (17). Briefly, mice were injected s.c. for 4 consecutive days with a single dose of 100 µg of recombinant murine granulocyte-CSF (G-CSF) (donated by Amgen, Thousand Oaks, CA) per kilogram of body weight, and on the next day, heparin-anticoagulated blood was collected. Human breast carcinoma SK-BR-3 cells, expressing HER2/neu (obtained from American Type Culture Collection, Mannassas, VA), were radioactively labeled by incubation of 1 x 106 cells with 150 µCi of 51Cr for 2 h at 37°C. After extensive washing, 51Cr-labeled SK-BR-3 cells were suspended at a concentration of 1 x 105 cells/ml. Aliquots of 50-µl suspensions of labeled SK-BR-3 cells were mixed with an equal volume of whole blood and a solution of 100 µl containing 10 µg/ml mAb. The mixed cell suspensions were incubated for 4 h at 37°C. The percentage of lysis of the SK-BR-3 cells was determined by measuring the radioactivity of the supernatants (17).
Passive cutaneous anaphylaxis
The method to determine passive cutaneous anaphylaxis was performed as described (8, 18), with modifications. Mice were injected intradermally at the basolateral side with various concentrations, ranging from 0.3 to 30 µg per mouse, of each of the following mAb: H5 (IgG1 anti-TNP), 7B4 (Ig2a anti-TNP), or 1B4 (IgG2b anti-TNP) (25 µl/injection spot). Two hours later, the mice were injected i.v. with 100 µl of a solution of physiologic saline containing 5 mg/ml HSA-TNP (Sigma) and 1% Evans blue (Sigma). Twenty minutes thereafter, skin sections of the mice were prepared, and a positive reaction was scored visually by determining blue staining of the injection spots due to extravasation of Evans blue.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Binding and phagocytosis of TNP-coated SRBC (SRBC-TNP), which were
opsonized with various murine anti-TNP mAb of different IgG
subclasses, by adherent thioglycolate-elicited macrophages were
studied. Opsonization of SRBC-TNP with each of two different IgG1 mAb,
two IgG2a mAb, and one IgG2b anti-TNP mAb resulted in efficient
binding (Fig. 1
A) and
phagocytosis (Fig. 1B) by macrophages from wild-type mice.
When macrophages from FcRIII-/- mice were used,
binding and phagocytosis of IgG2a or IgG2b-opsonized SRBC-TNP were
within the same range (Fig. 1, A and B). In sharp
contrast, both binding and phagocytosis of SRBC-TNP, which were
opsonized with each of both IgG1 mAb, were strongly reduced when using
FcRIII-/- macrophages (Fig. 1, A and
B). To confirm these results using a different panel of mAb
and macrophages in suspension, we tested the ability of murine IgG1,
IgG2a, and IgG2b mAb, directed against human glycophorin A, to induce
FcR-dependent rosette formation between HRBC and macrophages. The
majority (more than 85%) of wild-type macrophages formed rosettes with
IgG1, IgG2a, or IgG2b mAb-opsonized HRBC (Fig. 1C). HRBC
opsonized with IgG2a or IgG2b formed rosettes with macrophages from
FcRIII-/- mice equally well (Fig. 1C).
Rosette formation between IgG1-opsonized HRBC and macrophages from
FcRIII-/- mice was reduced almost to background level
(Fig. 1C). Together, these results demonstrate that
phagocytosis of IgG1-, but not IgG2a- or IgG2b-coated particles is
dependent on the presence of FcRIII, and indicate that the apparent
specificity of IgG1 toward FcRIII also occurs at the level of ligand
binding.
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Phagocytosis and intracellular degradation of endogenous
erythrocytes triggered by anti-erythrocyte Abs is one of the main
steps in the pathogenesis of autoimmune hemolytic anemia (AIHA) (19).
The involvement of FcR in the development of AIHA has been shown
previously using FcR -chain-deficient mice, which lack FcRI,
FcRIII, and FcRI (20). Hemolytic anemia through
erythrophagocytosis can be induced experimentally in mice by i.v.
injection of murine anti-murine erythrocyte mAb 105-2H (IgG1) or
34-4C (IgG2a) (14), both of which react with the same autoantigen
epitope (21). Two days after injection of wild-type mice with each of
these mAb, a pronounced uptake of endogenous erythrocytes by
macrophages in the liver was observed. When FcRIII-/-
mice were injected with anti-erythrocyte IgG1, no intracellular
erythrocytes could be detected in liver macrophages (Fig. 2). In contrast, after injection of
IgG2a, a significant, although partially reduced, phagocytosis of
erythrocytes by FcRIII-/- liver macrophages was
observed (Fig. 2). These results indicate that in vivo
erythrophagocytosis induced by autoreactive anti-erythrocyte IgG1
Abs is dependent on FcRIII.
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Previous studies demonstrated that trapping of immune complexes by
FDC, located in peripheral lymphoid tissues, occurs via complement and
Fc receptors (15, 16; reviewed in Ref. 22), and that Fc-mediated
trapping can be completely blocked by the anti-FcRII/III mAb
2.4G2. In the present study, to analyze the trapping capacity for
different IgG subclasses, complexes were prepared using murine
anti-TNP IgG mAb and TNP-PO. Complexes containing IgG1, IgG2a, or
IgG2b were efficiently trapped by FDC located in mesenteric lymph nodes
from wild-type mice. In sharp contrast, trapping of IgG1 complexes by
FDC in lymph nodes from FcRIII-/- mice was absent
(Fig. 3). Trapping of IgG2a or IgG2b
complexes by FDC in lymph nodes from either wild-type or
FcRIII-/- mice was within the same range (Fig. 3).
Immunocytochemistry revealed that staining with 2.4G2 of FDC in lymph
nodes from both wild-type and FcRIII-/- mice was
similar (data not shown). This provides evidence for expression of
FcRII on FDC, supporting the earlier proposed role of FcRII in
the deposition of immune complexes on FDC in germinal centers (23). In
the spleens of FcRIII-/- mice, trapping of IgG1
complexes was reduced significantly, while trapping of IgG2a or IgG2b
complexes was similar, when compared with spleens from wild-type mice
(data not shown). As a negative control, sections of lymph nodes or
spleens from wild-type or FcRIII-/- mice were
incubated with TNP-PO without IgG, which did not result in positive
staining (data not shown). Together, these results show that trapping
of complexed IgG1 by FDC in mesenteric lymph nodes is predominantly
mediated by FcRIII.
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Next, the functional consequences of the specific interaction
between IgG1 and FcRIII for neutrophil-mediated ADCC were studied,
using a previously described whole blood assay (17). Whole blood of
wild-type mice, which were treated with G-CSF, efficiently lysed
HER2/neu-expressing SK-BR-3 cells in the presence of each of
two different IgG1 or one IgG2a anti-HER2/neu mAb (Fig. 4). The effect of G-CSF on the lytic
activity of whole blood is due to an increase in the number of
circulating neutrophils, the main cytotoxic effector cell (17, 24).
This was confirmed by the observation that whole blood of wild-type
mice, which had not been treated with G-CSF, was not capable of lysing
Ab-coated SK-BR-3 cells (data not shown). In the absence of
anti-HER2/neu mAb, no lysis by blood from G-CSF-treated
mice was observed (Fig. 4), confirming it was Ab dependent. Strikingly,
no significant lysis was observed when SK-BR-3 cells were incubated
with whole blood from G-CSF-treated FcRIII-/- mice in
the presence of each of the two IgG1 mAb (Fig. 4). In contrast, whole
blood from FcRIII-/- mice was capable of lysing tumor
cells when incubated with IgG2a Abs (Fig. 4). These results indicate
that IgG1- but not IgG2a-induced neutrophil-mediated cytotoxicity is
dependent on the presence of FcRIII.
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We next investigated the ability of anti-TNP mAb of different
IgG subclasses to induce FcRIII-mediated passive cutaneous
anaphylaxis. The main effector cell in anaphylactic reactions is
assumed to be the mast cell, although there is increasing evidence for
the involvement of other cells such as eosinophils or neutrophils (25, 26). We induced anaphylaxis by intradermal injection of the mAb,
followed by i.v. injection of HSA-TNP. In wild-type mice, both IgG1 and
IgG2b anti-TNP triggered a profound and dose-dependent anaphylactic
reaction within 20 min after injection of the Ag, while at the same
concentrations IgG2a had no effect (Fig. 5). This demonstrates that murine IgG1
and IgG2b are able to mediate passive cutaneous anaphylaxis, which is
consistent with earlier documented observations (27). When using
FcRIII-/- mice, neither IgG1 nor IgG2b, at either
concentration used, was able to induce a detectable reaction (Fig. 5).
As a positive control, IgE anti-TNP induced a similar anaphylactic
response in both wild-type and FcRIII-/- mice (not
shown). These results indicate that IgG1- and IgG2b-induced passive
cutaneous anaphylaxis are triggered exclusively by FcRIII.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R, emphasizing the contribution of the
interaction between IgG1 and FcRIII to effector functions of the
immune system. An experimental system using FcRIII-deficient mice
and panels of mAb of different IgG subclasses was exploited, allowing
the examination of such processes both at a cellular level and under
physiologic in vivo conditions. The results revealed that FcRIII is
required for various IgG1-mediated functions. The processes in which we
demonstrated the specificity of the interaction between IgG1 and
FcRIII to be essential were the following.
First, phagocytosis of IgG1-coated erythrocytes by macrophages in vitro
was completely dependent on the presence of FcRIII. This extends our
previous observation (8) with a larger series of different IgG1, IgG2a,
and IgG2b Abs. The finding that FcRIII is also required for optimal
binding of IgG1-coated erythrocytes to macrophages indicates that
impaired phagocytosis of IgG1-coated erythrocytes by
FcRIII-/- macrophages may be due to an initial defect
in binding. Evidence for a functional in vivo consequence of the
specificity of IgG1 for FcRIII, with respect to phagocytosis, was
provided by the finding that IgG1-mediated erythrophagocytosis by liver
macrophages in vivo proved completely dependent on the presence of
FcRIII. Autoantibody-mediated ingestion of endogenous erythrocytes
represents one of the main processes causing AIHA (19, 20). The lack of
IgG1-mediated erythrophagocytosis in FcRIII-/- mice
coincides with a strong reduction in
anemia,4) indicating that the
interaction between IgG1 and FcRIII contributes significantly to the
development of experimental AIHA.
The specificity of interaction between IgG1 and FcRIII was not
restricted to particulate immune complexes, since we found that
trapping of soluble IgG1 complexes by FDC in mesenteric lymph nodes was
also dependent on the presence of FcRIII. This is in agreement with
previous studies showing that both complement and Fc receptors are
involved in trapping of IgG complexes by FDC (22, 23). It remains to be
established to what extent recognition of IgG1-containing immune
complexes by FcRIII on FDC contributes to Ag presentation and the
development of an immune response. It may well be that retention of
IgG1-containing immune complexes by FcRIII on FDC, by providing a
stimulus for selective differentiation of plasma cells into memory B
cells (28), participates in memory generation during Th2-like
responses.
Studying the proposed specificity of IgG1 toward FcRIII in a
functional assay determining Ab-dependent cytotoxicity, we observed
that FcRIII is absolutely required for IgG1-, but not IgG2a-mediated
lysis of tumor target cells by whole blood from G-CSF-treated mice.
This finding provides direct evidence for the involvement of FcR in
ADCC activity by neutrophils, consistent with our recent data in a
lymphoma model (29). The present results support the concept of
antitumor immunotherapy based on targeting of FcR expressed on effector
cells, using bispecific Abs recognizing both FcR and a tumor Ag (1, 17).
Finally, we tested our hypothesis in an in vivo inflammatory model,
i.e., passive cutaneous anaphylaxis, using soluble IgG complexes as a
trigger. Previously, we have shown that polyclonal rabbit IgG was not
able to trigger passive cutaneous anaphylaxis in
FcRIII-/- mice (8). The present study, using purified
murine mAb of specific IgG subclasses, shows that FcRIII is
absolutely required for passive cutaneous anaphylaxis induced by murine
IgG1. This supports the role of mast cells, which can degranulate in
vitro upon triggering of FcRIII (8, 30), in IgG-mediated
anaphylaxis. The present results confirm and extend previous data
concerning systemic anaphylaxis, performed with an IgG1 Ab as a
trigger, in FcR -chain-deficient mice (31).
The dependence on FcRIII may not be absolute for all IgG1-mediated
immune effector functions. For example, in other IgG1-mediated immune
effector functions not addressed in the present study, complement
activation may also play an additional role. In addition, a slight
residual binding of IgG1-coated SRBC to FcRIII-/-
macrophages was observed; this may be caused by weak interaction of
IgG1 with other receptors expressed on macrophages. Evidence for a
possible minor role of FcRII is provided by preliminary experiments
showing that the slight residual binding of IgG1-opsonized erythrocytes
to FcRIII-/- macrophages could be blocked by the
anti-FcRII and anti-FcRIII Ab 2.4G2 (W. Hazenbos and
J. E. Gessner, unpublished observation). This is consistent with
the earlier reported decreased binding of IgG1-coated particles to
macrophages from nonobese diabetic mice, which are defective in
expression of FcRII (32). The involvement of this putative
IgG1-FcRII interaction in triggering of effector functions remains
unclear and may depend on the relative expression level of FcRII on
the effector cells involved.
IgG2a was able to trigger significant responses in the absence of
FcRIII, i.e., in vitro and in vivo phagocytosis of erythrocytes by
macrophages, trapping of immune complexes by FDC, and lysis of tumor
cells. This IgG subclass may predominantly act via FcR other than
FcRIII, most probably being the high affinity receptor FcRI. This
was supported by our observation that IgG2a was not able to induce
lysis of tumor cells when using whole blood from G-CSF-treated FcR
-chain-deficient mice (I. Heijnen and W. Hazenbos, unpublished
observation), which lack both FcRI and FcRIII (5).
FcRIII-/- macrophages were able to bind and ingest
IgG2b-opsonized erythrocytes, implying that IgG2b can mediate these
processes via at least FcRI and/or FcRII. Remarkably, IgG2b was
able to trigger passive cutaneous anaphylaxis, which was abolished in
FcRIII-/- mice, confirming the indispensable role of
FcRIII in this effector mechanism (8). Taken together, these two
observations indicate that IgG2b can trigger effector functions both
via FcRIII and via other FcR, in contrast to IgG1, which
triggered predominantly via FcRIII. Murine IgG2b may have a broad
specificity for FcR in general, which is supported by the observation
that this IgG subclass, in contrast to IgG1 and IgG2a, interacts well
with different human FcR classes (33).
The present results provide better insight in the important role of the
interaction between IgG1 and FcRIII in effector functions of the
immune system. Subclass specificity of immune responses is known to be
crucial for protection against pathogens or induction of inflammation.
For example, in a mouse hepatitis virus infection model,
nonneutralizing antiviral IgG2a Abs were shown to protect efficiently
against a lethal infection, while IgG2b had no protective effect (34).
In an infection model with the fungal pathogen Cryptococcus
neoformans in mice, IgG1 is more potent than other IgG isotype
switch variants in mediating phagocytosis by macrophages and protection
to a lethal challenge, which recently has been shown to be FcR
-chain dependent (35, 36). The affinity of IgG subclasses for
cellular receptors may thus be a key factor in the outcome of immune
responses. In humans, variation in interaction between specific IgG
isotypes and FcR due to receptor polymorphisms has recently been
established to have strong implications for susceptibility to
infections and autoimmune diseases (37, 38). Further unraveling of the
involvement of specific IgG-FcR interactions in immune effector
functions is essential for rational development of FcR-targeted
immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. S. Verbeek, Department of Immunology, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address:
3 Abbreviations used in this paper: ADCC, antibody-dependent cellular cytotoxicity; AIHA, autoimmune hemolytic anemia; FDC, follicular dendritic cell; G-CSF, granulocyte colony-stimulating factor; HRBC, human red blood cells; HSA, human serum albumin; TNP, trinitrophenol; TNP-PO, trinitrophenol-peroxidase.
4 D. Meyer, C. Schiller, J. Westermann, S. Izui, W. L. W. Hazenbos, J. S. Verbeek, R. E. Schmidt, and J. E. Gessner. FcRIII (CD16) deficient mice demonstrate IgG isotype-dependent protection to experimental autoimmune hemolytic anemia. Submitted for publication.
Received for publication March 5, 1998. Accepted for publication May 19, 1998.
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R-directed immunotherapies. Immunol. Today 128:127.
chain deletion results in pleiotropic effector cell defects. Cell 76:519.[Medline]
RII-deficient mice. Nature 379:346.[Medline]
RIII (CD16) deficient mice. Immunity 5:181.[Medline]
receptor I bispecific antibodies. J. Immunol. 159:5629.[Abstract]
-chain does not alter maturation of the B cell response. J. Immunol. 159:2116.RI x murine MHC class II bispecific antibody. Cancer Immunol. Immunother. 45:166.[Medline]
RIII, but not FcRII, trigger serotonin release in rat basophilic leukemia cells. J. Immunol. 149:1365.[Abstract]
RIII: assessment of the cardiopulmonary changes, mast cell degranulation, and death associated with active or IgE- or IgG-dependent passive anaphylaxis. J. Clin. Invest. 99:901.[Medline]
RII gene expression in macrophages of NOD mice: genetic linkage with up-regulation of IgG1 and IgG2b in serum. J. Immunol. 157:4707.[Abstract]
RIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. Invest. 100:1059.[Medline]
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 S. E. Strome, E. A. Sausville, and D. Mann A Mechanistic Perspective of Monoclonal Antibodies in Cancer Therapy Beyond Target-Related Effects Oncologist, September 1, 2007; 12(9): 1084 - 1095. [Abstract] [Full Text] [PDF]
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 H. S. Bandukwala, B. S. Clay, J. Tong, P. D. Mody, J. L. Cannon, R. A. Shilling, J. S. Verbeek, J. V. Weinstock, J. Solway, and A. I. Sperling Signaling through Fc{gamma}RIII is required for optimal T helper type (Th)2 responses and Th2-mediated airway inflammation J. Exp. Med., August 6, 2007; 204(8): 1875 - 1889. [Abstract] [Full Text] [PDF]
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M. Zhao, M. E. Trimbeger, N. Li, L. A. Diaz, S. D. Shapiro, and Z. Liu Role of FcRs in Animal Model of Autoimmune Bullous Pemphigoid. J. Immunol., September 1, 2006; 177(5): 3398 - 3405. [Abstract] [Full Text] [PDF]
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Q. Lin, Y. Xiu, Y. Jiang, H. Tsurui, K. Nakamura, S. Kodera, M. Ohtsuji, N. Ohtsuji, W. Shiroiwa, K. Tsukamoto, et al. Genetic Dissection of the Effects of Stimulatory and Inhibitory IgG Fc Receptors on Murine Lupus J. Immunol., August 1, 2006; 177(3): 1646 - 1654. [Abstract] [Full Text] [PDF]
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Y. Hamaguchi, Y. Xiu, K. Komura, F. Nimmerjahn, and T. F. Tedder Antibody isotype-specific engagement of Fc{gamma} receptors regulates B lymphocyte depletion during CD20 immunotherapy J. Exp. Med., March 20, 2006; 203(3): 743 - 753. [Abstract] [Full Text] [PDF]
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F. Woelbing, S. L. Kostka, K. Moelle, Y. Belkaid, C. Sunderkoetter, S. Verbeek, A. Waisman, A. P. Nigg, J. Knop, M. C. Udey, et al. Uptake of Leishmania major by dendritic cells is mediated by Fc{gamma} receptors and facilitates acquisition of protective immunity J. Exp. Med., January 23, 2006; 203(1): 177 - 188. [Abstract] [Full Text] [PDF]
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N. M. O'Brien-Simpson, R. D. Pathirana, R. A. Paolini, Y.-Y. Chen, P. D. Veith, V. Tam, N. Ally, R. N. Pike, and E. C. Reynolds An Immune Response Directed to Proteinase and Adhesin Functional Epitopes Protects against Porphyromonas gingivalis-Induced Periodontal Bone Loss J. Immunol., September 15, 2005; 175(6): 3980 - 3989. [Abstract] [Full Text] [PDF]
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J. Rivera and A. Casadevall Mouse Genetic Background Is a Major Determinant of Isotype-Related Differences for Antibody-Mediated Protective Efficacy against Cryptococcus neoformans J. Immunol., June 15, 2005; 174(12): 8017 - 8026. [Abstract] [Full Text] [PDF]
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S. L. Lambert, C. Y. Okada, and R. Levy TCR Vaccines against a Murine T Cell Lymphoma: A Primary Role for Antibodies of the IgG2c Class in Tumor Protection J. Immunol., January 15, 2004; 172(2): 929 - 936. [Abstract] [Full Text] [PDF]
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 P. van Lent, K. C. Nabbe, P. Boross, A. B. Blom, J. Roth, A. Holthuysen, A. Sloetjes, S. Verbeek, and W. van den Berg The Inhibitory Receptor Fc{gamma}RII Reduces Joint Inflammation and Destruction in Experimental Immune Complex-Mediated Arthritides Not Only by Inhibition of Fc{gamma}RI/III but Also by Efficient Clearance and Endocytosis of Immune Complexes Am. J. Pathol., November 1, 2003; 163(5): 1839 - 1848. [Abstract] [Full Text] [PDF]
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 H. H. van Ojik, L. Bevaart, C. E. Dahle, A. Bakker, M. J. H. Jansen, M. J. van Vugt, J. G. J. van de Winkel, and G. J. Weiner CpG-A and B Oligodeoxynucleotides Enhance the Efficacy of Antibody Therapy by Activating Different Effector Cell Populations Cancer Res., September 1, 2003; 63(17): 5595 - 5600. [Abstract] [Full Text] [PDF]
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R. M. Tarzi, K. A Davies, J. W. C. Claassens, J. S. Verbeek, M. J. Walport, and H. T. Cook Both Fc{gamma} Receptor I and Fc{gamma} Receptor III Mediate Disease in Accelerated Nephrotoxic Nephritis Am. J. Pathol., May 1, 2003; 162(5): 1677 - 1683. [Abstract] [Full Text] [PDF]
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C. Taube, A. Dakhama, Y.-H. Rha, K. Takeda, A. Joetham, J.-W. Park, A. Balhorn, T. Takai, K. R. Poch, J. A. Nick, et al. Transient Neutrophil Infiltration After Allergen Challenge Is Dependent on Specific Antibodies and Fc{gamma}III Receptors J. Immunol., April 15, 2003; 170(8): 4301 - 4309. [Abstract] [Full Text] [PDF]
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 B. Nieswandt, W. Bergmeier, V. Schulte, T. Takai, U. Baumann, R. E. Schmidt, H. Zirngibl, W. Bloch, and J. E. Gessner Targeting of platelet integrin {alpha}IIb{beta}3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory Fc{gamma}R Int. Immunol., March 1, 2003; 15(3): 341 - 349. [Abstract] [Full Text] [PDF]
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 M. B. Jordan, N. van Rooijen, S. Izui, J. Kappler, and P. Marrack Liposomal clodronate as a novel agent for treating autoimmune hemolytic anemia in a mouse model Blood, January 15, 2003; 101(2): 594 - 601. [Abstract] [Full Text] [PDF]
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 H. H. Radeke, I. Janssen-Graalfs, E. N. Sowa, N. Chouchakova, J. Skokowa, F. Loscher, R. E. Schmidt, P. Heeringa, and J. E. Gessner Opposite Regulation of Type II and III Receptors for Immunoglobulin G in Mouse Glomerular Mesangial Cells and in the Induction of Anti-glomerular Basement Membrane (GBM) Nephritis J. Biol. Chem., July 19, 2002; 277(30): 27535 - 27544. [Abstract] [Full Text] [PDF]
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S. A. da Silveira, S. Kikuchi, L. Fossati-Jimack, T. Moll, T. Saito, J. S. Verbeek, M. Botto, M. J. Walport, M. Carroll, and S. Izui Complement Activation Selectively Potentiates the Pathogenicity of the IgG2b and IgG3 Isotypes of a High Affinity Anti-Erythrocyte Autoantibody J. Exp. Med., March 11, 2002; 195(6): 665 - 672. [Abstract] [Full Text] [PDF]
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 D. L. Laskin, B. Weinberger, and J. D. Laskin Functional heterogeneity in liver and lung macrophages J. Leukoc. Biol., August 1, 2001; 70(2): 163 - 170. [Abstract] [Full Text] [PDF]
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M. Afanasyeva, Y. Wang, Z. Kaya, S. Park, M. J. Zilliox, B. H. Schofield, S. L. Hill, and N. R. Rose Experimental Autoimmune Myocarditis in A/J mice Is an Interleukin-4-Dependent Disease with a Th2 Phenotype Am. J. Pathol., July 1, 2001; 159(1): 193 - 203. [Abstract] [Full Text] [PDF]
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N. Chouchakova, J. Skokowa, U. Baumann, T. Tschernig, K. M. H. Philippens, B. Nieswandt, R. E. Schmidt, and J. E. Gessner Fc{{gamma}}RIII-Mediated Production of TNF-{{alpha}} Induces Immune Complex Alveolitis Independently of CXC Chemokine Generation J. Immunol., April 15, 2001; 166(8): 5193 - 5200. [Abstract] [Full Text] [PDF]
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N. M. O'Brien-Simpson, R. A. Paolini, and E. C. Reynolds RgpA-Kgp Peptide-Based Immunogens Provide Protection against Porphyromonas gingivalis Challenge in a Murine Lesion Model Infect. Immun., July 1, 2000; 68(7): 4055 - 4063. [Abstract] [Full Text] [PDF]
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N. Lendvai, X.-W. Qu, W. Hsueh, and A. Casadevall Mechanism for the Isotype Dependence of Antibody-Mediated Toxicity in Cryptococcus neoformans-Infected Mice J. Immunol., April 15, 2000; 164(8): 4367 - 4374. [Abstract] [Full Text] [PDF]
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L. Fossati-Jimack, A. Ioan-Facsinay, L. Reininger, Y. Chicheportiche, N. Watanabe, T. Saito, F. M. A. Hofhuis, J. E. Gessner, C. Schiller, R. E. Schmidt, et al. Markedly Different Pathogenicity of Four Immunoglobulin G Isotype-Switch Variants of an Antierythrocyte Autoantibody Is Based on Their Capacity to Interact In Vivo with the Low-Affinity Fc{gamma} Receptor III J. Exp. Med., April 10, 2000; 191(8): 1293 - 1302. [Abstract] [Full Text] [PDF]
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A. Alonso, Y. Bayon, M. Renedo, and M. S. Crespo Stimulation of Fc{gamma}R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving I{kappa}B-{alpha} degradation and formation of p50/p65 NF-{kappa}B/Rel complexes Int. Immunol., April 1, 2000; 12(4): 547 - 554. [Abstract] [Full Text] [PDF]
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S. Wernersson, M. C. I. Karlsson, J. Dahlstrom, R. Mattsson, J. S. Verbeek, and B. Heyman IgG-Mediated Enhancement of Antibody Responses Is Low in Fc Receptor {gamma} Chain-Deficient Mice and Increased in Fc{gamma}RII-Deficient Mice J. Immunol., July 15, 1999; 163(2): 618 - 622. [Abstract] [Full Text] [PDF]
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