By
From the * Department of Experimental Immunology and the Department of Pathology, Institute of
Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan; § Core Research for
Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST),
Tokyo 101-0062, Japan;
Second Department of Pathology, Okayama University Medical School,
Okayama 700-8558, Japan; and ¶ Laboratory of Molecular Genetics and Immunology, The
Rockefeller University, New York 10021
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Abstract |
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It is widely accepted that immunoglobulin (Ig)E triggers immediate hypersensitivity responses
by activating a cognate high-affinity receptor, FcRI, leading to mast cell degranulation with
release of vasoactive and proinflammatory mediators. This apparent specificity, however, is
complicated by the ability of IgE to bind with low affinity to Fc receptors for IgG, Fc
RII and
III. We have addressed the in vivo significance of this interaction by studying IgE-mediated passive systemic anaphylaxis in Fc
R-deficient mice. Mice deficient in the inhibitory receptor
for IgG, Fc
RIIB, display enhanced IgE-mediated anaphylactic responses, whereas mice deficient in an IgG activation receptor, Fc
RIII, display a corresponding attenuation of IgE-mediated responses. Thus, in addition to modulating IgG-triggered hypersensitivity responses, Fc
RII
and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of
a regulatory pathway for IgE triggering of effector cells through IgG Fc receptors that could
contribute to the etiology of the atopic response.
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Introduction |
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The anaphylaxis reaction in mice has been considered to
be a typical immediate hypersensitivity response determined primarily by the activation of mast cells via antigen-induced aggregation of an IgE-sensitized high-affinity receptor for IgE (FcRI),1 causing the release of potent systemic
mediators (1, 2). The central role of Fc
RI in mediating the
response was demonstrated by observations that mice deficient in this receptor fail to undergo IgE-dependent, passive
cutaneous (3) and passive systemic anaphylaxis (4). These results were interpreted as indicating a necessary and sufficient
role for Fc
RI in mediating the IgE-dependent anaphylactic response, excluding the possibility for involvement of other
potential receptors for IgE (5). However, earlier observations indicated that the low-affinity Fc receptors for IgG
(Fc
RIIB and Fc
RIII) on mouse mast cells, macrophages,
and the rat mucosal type mast cell RBL-2H3 can bind IgE
immune complexes in vitro (6, 7), and the engagement of
Fc
RIIB/III with IgE immune complexes triggers C57.1
mast cells to release serotonin (6), suggesting a greater potential complexity to the IgE-mediated anaphylactic response.
Studies on active anaphylaxis in gene-targeted mice further challenged the simple model of IgE and FcRI as the
sole initiators of anaphylaxis and revealed a critical role for
IgG and Fc
R in this response. Induction of active anaphylaxis in mice deficient in IgE indicated that IgE antibodies
were not essential for the expression of systemic anaphylaxis
(8). In addition, mice deficient in Fc
RI mounted an undiminished active systemic anaphylactic response, whereas active sensitization and challenge of animals deficient in the
common
chain (FcR
/
) resulted in protection (9, 10).
Further support for the conclusion that type I immediate
hypersensitivity has a significant dependence on IgG1 and
Fc
Rs came from studies demonstrating that Fc
RIIB-deficient (Fc
RIIB
/
) mice exhibited an enhanced reaction in IgG1-mediated passive cutaneous anaphylaxis, thereby
establishing the importance of Fc
RIIB as an inhibitory
receptor under physiologic conditions (11), as suggested
previously in extensive in vitro studies by Daëron and colleagues (12, 13; for review see reference 14).
Although the evidence supporting a direct role for IgG
and FcRs in the anaphylaxis reaction is compelling, the
contribution of these receptors to the canonical IgE-mediated response is generally considered to be minimal. To directly analyze the roles of Fc
RIIB and Fc
RIII in the
IgE-dependent component of the systemic anaphylaxis reaction, we compared the responses elicited in Fc
RIIB
/
and Fc
RIII
/
mice upon passive transfer of either anti-TNP IgE or IgG followed by intravenous challenge with
TNP-OVA. As expected, Fc
RIIB
/
and Fc
RIII
/
mice
displayed enhanced or attenuated systemic anaphylaxis to IgG1 sensitization, respectively. However, contrary to the
accepted dogma, intense modulation of IgE-dependent systemic anaphylaxis was also observed in these Fc
R
/
mice
as a result of the low-affinity interactions of IgE-antigen complexes with these receptors. These studies demonstrate
the in vivo physiological significance of low-affinity IgE interactions with Fc
Rs and represent a novel regulatory pathway for classical type I hypersensitivity responses.
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Materials and Methods |
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Antibodies.
Rat anti-mouse FcAnimals.
All experiments were performed on 6-12-wk-old mice. Male and female FcInduction of Passive Systemic Anaphylaxis.
Mouse IgG1 or IgE anti-TNP mAbs were administered intravenously through the tail vein in volumes of ~200 µl/mouse. 30 min after injection of anti-TNP IgG1 or 24 h after injection of IgE, mice were injected with 1.0 mg i.v. TNP4-OVA in PBS. Control mice received OVA in PBS instead. The concentration of IgG1 and IgE mAbs used for passive sensitization and the amount of TNP-OVA used for challenge was determined based on preliminary dose-response experiments required to produce significant drops in body temperature in wild-type and FcMonitoring of Rectal Temperature and Heart Rate.
Changes in core body temperature associated with systemic anaphylaxis were monitored by measuring changes in rectal temperature using a rectal probe coupled to a digital thermometer (Natsume Seisakusyo Co.) as described (4, 9, 10). Heart rate was recorded as electrocardiograms (Nihon Kohden) of mice under 2,2,2-tribromoethanol (0.25 mg/g body weight, i.p.) anesthesia.Flow Cytometric Analysis.
Bone marrow-derived cultured mast cells (BMMC) were prepared as described previously (3). For monitoring of upregulation of FcELISA Determinations for Blood Histamine.
Blood was collected from subocular plexus of mice into microcentrifuge tubes containing EDTA on ice at 5 min after antigen challenge, and plasma was prepared. Histamine in the plasma samples was quantified using ELISA plates (ICN Pharmaceuticals, Inc.) according to the manufacturer's instructions.Histological Study.
Mice were killed by cervical dislocation. Their tissues were removed and fixed in 10% (vol/vol) neutral buffered formalin and then embedded in paraffin. The specimens were sectioned at 3 µm and stained with toluidine blue at pH 4.0. The number of mast cells/mm2 was determined under a light microscope. A `degranulated' mast cell was defined as a cell showing extrusion of >10% cell granules.Statistical Analysis.
Statistical differences were calculated using Student's t test or Fisher's test. P < 0.05 was considered significant. ![]() |
Results and Discussion |
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Bocek et al. (7) reported
that coclustering of FcRIIB and Fc
RIII on RBL-2H3
cells did not lead to stimulation of the cells, suggesting a possible inhibitory role of Fc
RIIB in this process. In addition,
in vitro observations by Daëron et al. (12) demonstrated that
mast cell secretory responses triggered by Fc
RI may be
controlled by Fc
RIIB/III. Moreover, the regulatory role of
Fc
RIIB was also observed in the cellular activation process via B cell receptors (19) and T cell receptors (13; for review see reference 14). Our previous studies using gene-targeted mice had demonstrated the role of Fc
RIIB in modulating IgG1-mediated passive cutaneous anaphylaxis (11). To
establish the generality of those in vivo observations, we investigated IgG1-mediated passive systemic anaphylaxis in
Fc
RIIB
/
and Fc
RIII
/
mice. We chose to evaluate a
passive rather than active model in our studies because
Fc
RIIB
/
mice display enhanced humoral immune responses (11) that could complicate the comparison and interpretation of the anaphylactic responses. To elicit the anaphylactic response, mice were injected intravenously with IgG1
specific for TNP, followed by intravenous administration of
TNP-OVA 30 min later. Fig. 1 A shows that Fc
RIIB
/
mice developed an enhanced IgG1-dependent passive systemic anaphylactic response as compared with passively sensitized wild-type controls challenged with TNP-OVA. In
wild-type mice, the decrease in core temperature was also
transient, reaching a nadir ~15 min after induction, whereas
the drop in temperature of Fc
RIIB
/
mice persisted for
more than 30 min without returning to baseline.
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The mAb 2.4G2 is specific for the extracellular domains of
murine FcRIIB and Fc
RIII (22). 2.4G2 induces a degranulative response in BMMC, which is enhanced in cells derived
from Fc
RIIB
/
mice (11). This enhancement is apparent
in vivo as well as shown in Fig. 1 B, where the decrease in
core temperature after administration of 2.4G2 was more pronounced in Fc
RIIB
/
mice than in control mice. These
results indicate that Fc
RIIB on effector cells, such as mast
cells, inhibits the systemic anaphylaxis elicited via Fc
RIII.
In contrast to the enhanced responses in Fc
RIIB
/
mice
described above (Fig. 1, A and B), both Fc
RIII
/
mice and
FcR
/
mice failed to develop IgG1-mediated passive
systemic anaphylaxis (Fig. 1 C), directly establishing that
IgG1-mediated anaphylaxis is triggered through Fc
RIII,
as was indirectly suggested by others (9, 10).
As IgE immune complexes can bind with low affinity to FcRII and III in vitro, we next induced passive
systemic anaphylaxis upon anti-TNP IgE adoptive transfer
and TNP-OVA administration into Fc
RIIB
/
mice.
IgE-mediated systemic anaphylaxis was significantly enhanced in Fc
RIIB
/
mice, as assessed by changes in core temperature (Fig. 2 A), heart rate (Fig. 2 B), and augmented
hemorrhage in the ileum villi (Fig. 2 C). These results indicate that IgE/Fc
RI-mediated anaphylaxis is facilitated by
the deletion of Fc
RIIB in vivo without any apparent involvement of IgG-immune complexes.
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Systemic anaphylaxis can result in a fatal outcome. In
mice, this mortality has been shown to be associated with
IgG1 and FcRIII (9). As shown in Table I, we observed
mortality as a consequence of the anaphylactic response
only in Fc
RIIB
/
mice upon administration of either
IgG1 or IgE and the corresponding antigen, or 2.4G2.
These results confirm that either IgE- or IgG-induced systemic anaphylaxis is indeed augmented in Fc
RIIB
/
mice, as assessed by mortality during anaphylaxis.
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These unexpected observations for IgE-mediated anaphylaxis prompted us to examine
whether deletion of FcRIIB influenced Fc
RI expression
levels on effector cells. We confirmed by flow cytometric analysis that the expression level of Fc
RI on BMMC from
Fc
RIIB
/
mice was comparable to the level on wild-type BMMC (data not shown). In addition, we could not
demonstrate any significant difference in the expression
levels of Fc
RI on mast cells after IgE-induced upregulation in vitro or in vivo (Fig. 3, A and B). As shown in Fig.
3 A, BMMC derived from either from Fc
RIIB
/
or
wild-type mice displayed the same level of upregulation of Fc
RI in response to IgE (18). Similarly, peritoneal mast
cells isolated from Fc
RIIB
/
and wild-type mice 24 h
after intravenous administration of 20 µg IgE had equivalent levels of Fc
RI (Fig. 3 B). Histopathological examinations indicated that the density and morphology of mast cells in ear, abdominal skin, and trachea from the mutant
mice were not significantly different from those in wild-type mice (data not shown).
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The mechanism by which FcRIIB
/
mice augmented IgE-mediated anaphylaxis was examined by determining the activation of effector cells in these animals as
compared with their wild-type counterparts. Blood histamine levels were measured after the induction of anaphylaxis in Fc
RIIB
/
and wild-type mice. As shown in Fig.
4 A, blood obtained both from wild-type or Fc
RIIB
/
-sensitized animals 5 min after challenge with antigen or
2.4G2 revealed increased histamine concentrations. The
histamine levels seen in Fc
RIIB
/
-challenged mice were
consistently higher in response to IgE, IgG1, or 2.4G2
stimulation than in control mice, suggesting that the enhanced anaphylaxis in Fc
RIIB
/
mice could be interpreted in part by accelerated activation of mast cells in the
mutant animals. To directly demonstrate enhanced degranulation, lung samples from Fc
RIIB
/
or wild-type mice
were removed before and 30 min after the induction of
IgG-mediated passive systemic anaphylaxis and examined
histopathologically. As shown in Fig. 4 B and E, mast cells
around bronchi in Fc
RIIB
/
mice displayed quantitatively more degranulation than comparable samples taken
from wild-type mice subjected to similar treatment.
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Although Takizawa et al. (6) demonstrated
that FcRIIB and Fc
RIII act as low-affinity receptors for
IgE on cultured mast cells and macrophages in vitro, the
physiological significance of this interaction between IgE
and Fc
RIIB/III has not been established. The consequence
of a low-affinity interaction between IgE and Fc
Rs in
vivo would result in IgE immune complexes binding not only to Fc
RI but also to Fc
RIIB/III on those cells and
potentially modulating mediator release. Dombrowicz et al.
(4) have shown that although BMMC from Fc
RI
/
mice can bind IgE immune complexes via Fc
RIIB/III in
vitro, the abrogation of IgE-mediated systemic anaphylaxis
in vivo by deletion of Fc
RI would indicate that the interaction of IgE with Fc
Rs is not significant. However, an
alternative explanation for their data is suggested by the
present studies, as the Fc
RI
/
strain retains Fc
RIIB as
well as Fc
RIII on its mast cells (4). Based on our data, we
propose that the IgE immune complex-mediated response
would represent the sum of three components, i.e., an Fc
RI-mediated major positive factor, an Fc
RIIB negative response, and an Fc
RIII-mediated positive component, respectively. When the Fc
RI component had been
lost, the sum of the remaining Fc
RIIB and Fc
RIII components would be negligible. Our present results predict
that a sum of the components of Fc
RI and Fc
RIIB
would be a positive, although diminished, response. This
prediction is supported by the IgE-mediated anaphylactic
response in Fc
RIII
/
mice. As shown in Fig. 5 A,
Fc
RIII
/
mice indeed show a decreased response in IgE-mediated systemic anaphylaxis. Moreover, we found that
blocking of Fc
RIIB by preadministration of 2.4G2 resulted in an enhanced response in IgE-mediated systemic anaphylaxis in Fc
RIII
/
mice (Fig. 5 B). Taken together,
these results support the conclusion that Fc
RIIB attenuates IgE-mediated anaphylactic responses triggered by Fc
RI
or Fc
RIII.
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Further support for the role of FcRIIB in modulating
the IgE-mediated response comes from studies in Src homology 2-containing inositol phosphatase (SHIP)-deficient
mice (23). This inositol polyphosphate phosphatase is recruited to Fc
RIIB upon cross-linking with an immunoreceptor tyrosine-based activation motif (ITAM)-containing activation receptor through its SH2 (Src homology 2) domain and leads to the hydrolysis of phosphatidylinositol
3,4,5-trisphosphate, with release of Bruton's tyrosine kinase
and phospholipase C
from the inner leaflet of the cell membrane (24). The net result of this pathway is the termination
of calcium influx, with subsequent inhibition of activation responses (20, 21, 25). Mast cells derived from SHIP-deficient
mice display a hyperresponsive IgE phenotype similar to the
response seen in Fc
RIIB
/
mice (26). Thus, functional uncoupling of Fc
RIIB from its signaling pathway results in
similar phenotype deletion of the receptor itself.
The observations presented here support the hypothesis
that IgE-mediated activation is modulated by inhibitory receptors like FcRIIB. Perturbation of an inhibitory pathway would be predicted to render mast cells more sensitive
to IgE activation and could account for some atopic phenotypes. Upregulation of Fc
RIIB or its constitutive engagement would result in desensitization of mast cells to IgE
triggering and reversal of the atopic state.
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Footnotes |
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Address correspondence to Toshiyuki Takai, Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo, Sendai 980-8575, Japan. Phone: 81-22-717-8501; Fax: 81-22-717-8505; E-mail: tostakai{at}idac.tohoku.ac.jp
Received for publication 25 January 1999 and in revised form 5 March 1999.
The authors wish to thank Dr. Yutaka Kagaya, Department of Internal Medicine, Tohoku University, Japan, for his assistance in the measurement of heart rate and for helpful discussions.
This work was supported by research grants from the Ministry of Education, Science, Sports, and Culture of Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST) (to T. Takai); and from the National Institutes of Health and the Juvenile Diabetes Foundation (to J.V. Ravetch).
Abbreviations used in this paper
BMMC, bone marrow-derived cultured
mast cells;
FcRI, high-affinity receptor for IgE;
Fc
R, Fc receptor for
IgG;
FcR
, Fc receptor
subunit;
Fc
RIIB and Fc
RIII, type IIB and
type III low-affinity receptors for IgG, respectively.
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