By
From the * Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, Massachusetts 02215; Division of Immunology, Children's Hospital and Harvard
Medical School, Boston, Massachusetts 02215; and the § Departments of Pediatrics and Medicine,
Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda,
Maryland 20814
The binding of immunoglobulin E (IgE) to high affinity IgE receptors (FcRI) expressed on
the surface of mast cells primes these cells to secrete, upon subsequent exposure to specific antigen, a panel of proinflammatory mediators, which includes cytokines that can also have immunoregulatory activities. This IgE- and antigen-specific mast cell activation and mediator
production is thought to be critical to the pathogenesis of allergic disorders, such as anaphylaxis
and asthma, and also contributes to host defense against parasites. We now report that exposure
to IgE results in a striking (up to 32-fold) upregulation of surface expression of Fc
RI on
mouse mast cells in vitro or in vivo. Moreover, baseline levels of Fc
RI expression on peritoneal mast cells from genetically IgE-deficient (IgE
/
) mice are dramatically reduced (by
~83%) compared with those on cells from the corresponding normal mice. In vitro studies indicate that the IgE-dependent upregulation of mouse mast cell Fc
RI expression has two components: an early cycloheximide-insensitive phase, followed by a later and more sustained component that is highly sensitive to inhibition by cycloheximide. In turn, IgE-dependent
upregulation of Fc
RI expression significantly enhances the ability of mouse mast cells to release serotonin, interleukin-6 (IL-6), and IL-4 in response to challenge with IgE and specific
antigen. The demonstration that IgE-dependent enhancement of mast cell Fc
RI expression
permits mast cells to respond to antigen challenge with increased production of proinflammatory and immunoregulatory mediators provides new insights into both the pathogenesis of allergic diseases and the regulation of protective host responses to parasites.
Mast cells are widely distributed in vascularized tissues
and serosal cavities, where they can function as effector cells in IgE-dependent immune responses (1). Indeed, IgE- and antigen-dependent activation of mast cell
mediator secretion is thought to contribute significantly to
both the pathogenesis of allergic diseases (1, 4) and the
expression of acquired immunity to infection with certain
parasites (1, 3).
Several lines of evidence, including the phenotype of
mice genetically engineered to lack expression of either the
Fc There have been no reports that circulating levels of IgE
can influence the expression of Fc In this report, we used in vitro, in vivo, and genetic approaches to examine whether IgE can directly regulate Fc Flow Cytometry of Mouse Peritoneal Mast Cells and In Vitro-derived
Mouse Mast Cells.
In the mouse, mast cells express two surface
receptors that can bind IgE, the high affinity receptor that binds
IgE monomers, FcRI
chain (9) or the FcR
chain (10), indicate that
mast cells (and basophils, circulating granulocytes that can
produce many of the same mediators as mast cells; references 1) must display high affinity IgE receptors (Fc
RI)
on their surface in order to express significant IgE- and antigen-specific effector function. Yet the factors that regulate the expression of Fc
RI on the surface of these effector
cells are incompletely understood (5, 6). Two groups independently demonstrated that the level of Fc
RI expression
on circulating human basophils can exhibit a positive correlation with the serum concentration of IgE (11).
However, the basis for this association was not determined.
Malveaux et al. (13) proposed two possible explanations for
their findings, i.e., a genetic association between serum IgE
and the number of basophil IgE receptors or, more likely,
in their view, the modulation of basophil receptor number
by the serum IgE concentration. Another possibility is that
some common factor (or factors), such as immunoregulatory cytokines, can influence both IgE levels and basophil
Fc
RI expression.
RI on mast cells. However, two studies demonstrated that short-term incubation
of the rat basophilic leukemia cell line (RBL-2H3) with IgE
in vitro can result roughly in a doubling of the cells' surface
expression of Fc
RI (14, 15). Based on an analysis of several
of its phenotypic characteristics, the RBL-2H3 cell line probably should be regarded as of mast cell, rather than basophil, origin (16). The findings of Furuichi et al. (14) and
Quarto et al. (15) thus indicate that exposure to monomeric IgE in vitro can result in a modest increase in Fc
RI expression in a long-term malignant mast cell line. These
studies also showed that this effect, which was insensitive to
inhibition by cycloheximide (14), probably largely reflected
IgE-dependent suppression of the elimination of Fc
RI
from the cell surface (14, 15). However, the relevance of
these observations to normal mast cells was not clear. More
importantly, it was not established whether IgE-induced
increases in Fc
RI expression resulted in enhanced responsiveness to IgE-dependent release of proinflammatory mediators.
RI
expression on the surface of mouse mast cells. We found
that IgE can substantially upregulate Fc
RI expression on
mouse mast cells in vitro or in vivo, and showed that this
IgE-dependent enhancement of mouse mast cell Fc
RI expression, in turn, can significantly increase the ability of
these cells to release proinflammatory and immunoregulatory mediators in response to IgE-dependent activation.
RI, and Fc
RII/III, which can bind IgE immune complexes (17, 18). By contrast, the low affinity IgE receptor, CD23, appears to be restricted to cells other than mast cells
(e.g., B cells) in the mouse (18, 19). Because our studies employed monomeric IgE, IgE binding to mouse mast cells under
the conditions of our experiments reflects largely, if not entirely,
binding of IgE to Fc
RI (18). Nevertheless, for flow cytometric
analysis of unfractionated freshly isolated mouse peritoneal cells (20), we preincubated the cells with B3B4 (21) and 2.4G2 (18, 22) mAbs for 15 min to block low affinity binding of IgE or other subsequent antibodies to CD23 (on B cells, reference 19) or Fc
RII/III (on mast cells or monocytes/macrophages, references 18,
22), respectively. The cells were then incubated at 4°C with a
mouse IgE anti-DNP mAb (reference 23, 10 µg/ml) for 50 min,
to saturate fully any Fc
RI expressed by these cells (18), and then,
simultaneously, for the last 25 min, with a biotinylated rat anti-
mouse c-kit antibody (PharMingen, San Diego, CA, at 15 µg/ml).
After washing once in DMEM (GIBCO BRL, Gaithersberg, MD)
supplemented with 5% FCS (Sigma Chemical Co., St. Louis,
MO), cells were stained with FITC-rat anti-mouse IgE antibody
(PharMingen, at 10 µg/ml) and PE-streptavidin (Sigma Chemical Co., at 14 µg/ml) for 25 min at 4°C. Stained cells were analyzed using a FACSCalibur® (Becton Dickinson, San Jose, CA).
Autofluorescent cells (primarily macrophages) were rejected to
identify mast cells (IgE+, c-kit+) clearly. 20,000 peritoneal cells in
each sample were analyzed, and at least 100 mast cells were studied to calculate the median value of fluorescence intensity. We
confirmed that the gated IgE+, c-kit+ cells were exclusively mast
cells by staining cytocentrifuge preparations of the sorted cells
with May-Grünwald-Giemsa stain and then examining them by
light microscopy at ×400.
Western Blot Analysis of FcRI
Chain in Mouse Mast Cells.
For Western blot analysis, 2.5 × 107 of the same BMCMCs used
for flow cytometry (i.e., just before analysis, the cells were incubated with excess IgE at 4°C to saturate their Fc
RI, see above)
were extracted in 0.2 M borate buffer containing 0.5% Triton X-100
and proteinase inhibitors (26) and postnuclear extracts were subjected to immunoprecipitation with noncoated beads (preclear) and
then two times with anti-IgE-coated beads (10 µg affinity-purified rabbit anti-IgE per precipitation), to isolate surface-expressed Fc
RI that had bound IgE. Immunoprecipitates were pooled, resolved by 10% SDS-PAGE under reducing conditions, and immunoblotted to Immubilon P (Millipore, Bedford, MA). No antibodies suitable for selectively identifying the mouse Fc
RI
chain have been described. Accordingly, Western blotting was
performed with the JRK anti-mouse Fc
RI
chain mAb (26).
Quantification of Mouse Mast Cell Mediator Release. For measurements of serotonin (5-hydroxytryptamine, 5-HT) release, BMCMCs (>95% purity) or peritoneal cells were incubated with ascites IgE at 10 µg/ml and [3H]5-HT (NEN, Boston, MA) at 2 µCi/ml for 2 h at 37°C, washed, and 5-HT serotonin release was calculated as the percentage of total incorporated [3H]5-HT present in the cell supernatant 10 min after stimulation with DNP human serum albumin (DNP-HSA) (Sigma), the calcium ionophore A23187 (Sigma), or vehicle at 37°C (27). For IL-6 or IL-4 release, BMCMCs were sensitized with ascites IgE at 10 µg/ml for 50 min at 4°C, and stimulated with DNP-HSA for 4 h at 37°C. IL-6 or IL-4 in the supernatants was measured using ELISA kits (Endogen, Cambridge, MA).
Assessment of the Effect of Administration of IgE In Vivo on Mouse
Mast Cell FcRI Expression.
12-22-wk-old mice were used in
four separate in vivo experiments, two with BALB/c mice (data
combined in Fig. 7 A), one with untreated IgE
/
and corresponding IgE +/+ mice (Fig. 7 B), and one with antibodytreated IgE
/
mice (Fig. 7 C). Purified mouse IgG2a mAb
(Sigma, clone UPC 10) and mouse anti-DNP IgE mAb (Sigma, clone SPE-7) were dialyzed to remove NaN3, centrifuged at
100,000 g for 1 h at 4°C, and filtered before use. For i.v. treatment, IgG2a or IgE (100 µg in 0.24 ml PBS) or 0.24 ml of PBS
alone was administered to each mouse daily by tail vein. Blood
was obtained at time of death (~18 h after the last i.v. injection)
for measurement of total serum IgE concentration by ELISA (28).
For assay of serotonin release, peritoneal cells were incubated for
20 h in DMEM containing 10% FCS (27) and then sensitized
with IgE, labeled with [3H]5-HT, and stimulated with DNP-
HSA as described above.
Statistics. Unless otherwise specified, all data are expressed as mean ± SEM, and all differences between values were compared by the two-tailed Student's t test (unpaired).
As assessed by flow cytometry to detect
binding of IgE (see Materials and Methods), we found that
the baseline levels of FcRI expression on the surface of
BALB/c mouse peritoneal mast cells, identified as an IgE+,
c-kit+ subpopulation of unfractionated peritoneal cells (Fig. 1
A), were greatly enhanced, in a concentration- (Fig. 1, B and
C) and time- (Fig. 1 D) dependent manner, upon incubation
of the cells in vitro with a mouse monoclonal IgE antibody.
Moreover, Fc
RI expression progressively diminished when
peritoneal mast cells were incubated in vitro without added
IgE (Fig. 1 D). As a result, levels of Fc
RI expression in
peritoneal mast cells incubated for 6 d with IgE at 5 µg/ml
(i.e., in the range observed in the serum of parasite-infected
mice, reference 28) were 20-fold higher than those in cells
incubated for 6 d without IgE (Fig. 1 D). And mast cells incubated for 4 d with a concentration of IgE (0.05 µg/ml)
comparable to that present in the serum of normal mice
(28, 29) had levels of Fc
RI expression that were 3.6-fold those in cells incubated for 4 d without IgE (Fig. 1 C).
IgE Enhances Fc
Unfractionated peritoneal cells contain cells other than mast cells, some
of which may have influenced the effect of IgE on peritoneal mast cell FcRI expression. Therefore, we next assessed whether IgE also enhanced surface expression of Fc
RI in
essentially pure populations of immature mouse mast cells
derived in vitro from the bone marrow cells of IgE null
mice (IgE
/
mice) or the corresponding normal (IgE
+/+) BALB/c mice (24). Incubation with IgE (at 5 µg/ml)
resulted in a marked elevation (32-fold at day 4) in Fc
RI
expression in BMCMCs from IgE +/+ mice (Fig. 2 A).
Essentially identical results were obtained in two additional experiments with BMCMCs derived from IgE
/
mice
(data not shown), cell preparations which could not have
contained any source of endogenous mouse IgE (i.e., small
numbers of contaminating B cells).
Studies of cells transfected with either mouse FcRI, Fc
RII, or Fc
RIII indicate that washing of the cells prior to
flow cytometric analysis does not significantly remove IgE
from Fc
RI, but can largely eliminate IgE or IgG from Fc
RII/Fc
RIII (18). Accordingly, under the conditions of our
experiments, the IgE binding that we assessed by flow cytometry reflected largely, if not entirely, the binding of IgE to
Fc
RI. Nevertheless, we used Western blotting to assess directly the extent to which the
chain of the Fc
RI was associated with the BMCMC surface receptors that bound IgE
in our experiments. We found that the increase in IgE binding
that was detected by flow cytometry after incubation of
BMCMCs with IgE (at 5.0 µg/ml) for 21 h (~14-fold increase versus BMCMCs that had not been incubated with
IgE) directly paralleled the amount of Fc
RI
chain obtained
from anti-IgE immunoprecipitates of these cells (an increase of
~17-fold versus control BMCMCs that had not been incubated with IgE) (Fig. 2 B). These data provide further evidence
that the increased IgE-binding ability, which we detected on
the surface of mast cells that had been incubated with IgE,
indeed reflected increased surface expression of Fc
RI.
The mast cells that were incubated with the highest concentration of ascites IgE that was used routinely in our experiments (i.e., 5 µg IgE/ml) were exposed to an ~100-fold
dilution of the stock preparation of ascites. However, this
ascites-derived IgE preparation also significantly enhanced
mast cell FcRI expression even when tested at dilutions of
10,000- to 100,000-fold (to yield IgE concentrations of
0.05 or 0.005 µg/ml, respectively, e.g., see Fig. 1 C). Nevertheless, to rule out the possibility that some constituent of
the ascites preparation other than IgE might have significantly influenced our results, we incubated aliquots of the
same population of BALB/c BMCMCs for 4 d with either
ascites or affinity-purified IgE at 5 µg IgE/ml. We found
that either preparation of IgE markedly increased the
Fc
RI expression of the cells, and to essentially equivalent
levels (Fig. 3 A). Similar results were obtained in two additional experiments. By contrast, incubation of such BMCMCs
for 4 d with a mouse IgG2a mAb at 5 µg/ml had no detectable effect on the cells' surface expression of Fc
RI
(Fig. 3 A). A separate experiment gave the same result.
We also tested the effects of a 4-d incubation of BALB/c
BMCMCs with IgG2a at 5-1000 µg/ml. As expected, we
found that incubation with ascites IgE at 5 µg/ml markedly
increased BMCMC expression of FcRI (Fig. 3 B), whereas
either affinity-purified IgG2a, at 5 or 100 µg/ml (data not
shown) or 200 µg/ml (Fig. 3 B), or ascites IgG2a, at 5 or
100 µg/ml (data not shown) or 500 or 1,000 µg/ml (Fig. 3
C), had little or no effect on Fc
RI expression.
The effect of IgE on BMCMC FcRI expression appeared to be specific, in that cells incubated for 4 d with
IgE at 5 µg/ml exhibited no detectable change in their surface expression of Fc
RII/III, as assessed by flow cytometry with the 2.4G2 antibody (Fig. 3 D). A 4-d incubation
of BMCMCs with purified IgG2a mAb (at 5 or 200 µg/
ml, data not shown) or ascites IgG2a mAb (at 1,000 µg/ml; Fig. 3 D) also had little or no effect on the cells' surface expression of Fc
RII/III, as assessed by flow cytometry.
To assess the functional significance of IgE-dependent upregulation of mast cell FcRI expression, we incubated BALB/c BMCMCs with 0, 0.005, or 5 µg/ml of IgE
for 4 d, then passively sensitized the cells with excess monoclonal anti-DNP IgE (10 µg/ml) for 2 h, and then challenged the cells with various concentrations of specific antigen (DNP-HSA) and measured release of the cytoplasmic granule-associated mediator serotonin (5-HT) and the cytokines, IL-6 and IL-4. As shown in Fig. 4, the IgE-induced
enhancement of Fc
RI expression (Fig. 4 A) was associated
with a significantly enhanced functional responsiveness to
antigen stimulation, as assessed either by 5-HT release (Fig.
4 B) or IL-6 (Fig. 4 C) or IL-4 (Fig. 4 D) production. The
results shown in Fig. 4 are representative of those obtained
in three other experiments in which we assessed 5-HT release and one other experiment in which we assessed IL-6
and IL-4 release.
Notably, IgE-dependent enhancement of mast cell FcRI
expression both decreased the concentration of antigen required to elicit IgE-dependent mediator release and markedly increased mediator secretion at a given antigen concentration (Fig. 4, B-D). For example, when challenged
with antigen at 30 ng/ml, BMCMCs that had been incubated for 4 d with IgE at 5.0 µg/ml exhibited ~60% greater specific release of 5-HT (Fig. 4 B) and ~6-fold
greater release of IL-6 (Fig. 4 C) than did mast cells which
had been incubated for 4 d with IgE at 0.005 µg/ml. The
effect on IL-4 production was especially striking, in that
the only cells which exhibited detectable release of IL-4
upon challenge with specific antigen were those which had
been incubated for 4 d with IgE at 5.0 µg/ml (Fig. 4 D).
The same finding was obtained in an additional experiment
with BMCMCs derived from IgE
/
mice (data not
shown).
The effect of a 4-d incubation with IgE on the ability of
BMCMCs to release mediators in response to challenge with
IgE and antigen did not reflect a general enhancement of
the secretory responsiveness of the cells, in that there was
no significant parallel increase in the ability of the cells to
release 5-HT in response to stimulation with the calcium
ionophore A23187 (Fig. 5). The results shown in Fig. 5 are
representative of those obtained in two separate experiments with BMCMCs and one with peritoneal mast cells.
The IgE-dependent Increase in Mast Cell Fc
In RBL cells, the ~50-90% increase in FcRI
surface expression that occurs as a result of incubation of
the cells with IgE for 4 h is insensitive to inhibition with
cycloheximide at 10 µg/ml (14). This, and other lines of
evidence, support the hypothesis that the modest change in
Fc
RI surface expression observed in this setting does not require protein synthesis but instead largely or entirely reflects
the reduced loss from the cell surface of Fc
RI that are occupied by IgE (14, 15). In confirmation of these observations
with rat basophilic leukemia (RBL) cells, we found that incubation of BMCMCs with cycloheximide (at 1, 3, or 10 µg/ml) had little or no effect on the roughly fourfold increase in Fc
RI surface expression that occurred in the first
3 h after incubation of the cells with IgE at 5 µg/ml (Fig. 6
A). However, cycloheximide treatment profoundly suppressed the subsequent, more sustained, increase in Fc
RI
expression by these cells (Fig. 6 A). Cycloheximide treatment also resulted in a significant reduction in surface expression of Fc
RI in BMCMCs that were maintained in
culture without added IgE (Fig. 6 A). Similar results were obtained in a second experiment.
Note that the cycloheximide-insensitive and cycloheximide-sensitive components of the IgE-induced enhanced
surface expression of FcRI were detectable at relatively
early intervals after addition of IgE (3 h or 6-12 h, respectively; Fig. 6 A), before the cells treated with cycloheximide began to exhibit substantially diminished viability, as
revealed by staining with Trypan blue (Fig. 6 B). Moreover, the effects of cycloheximide on Fc
RI surface expression were essentially identical in BMCMCs treated with the
drug at 1, 3, or 10 µg/ml (Fig. 6 A), whereas, as expected,
cells treated with higher concentrations of the agent exhibited more toxicity at late intervals of culture than did those
treated with cycloheximide at 1 µg/ml (Fig. 6 B).
We performed four experiments to investigate whether
IgE is an important regulator of mast cell FcRI expression
in vivo. In two experiments, affinity-purified mouse IgE
mAb (100 µg/day), or either affinity-purified mouse IgG2a
mAb (100 µg/day) or vehicle (PBS) alone, were administered
to BALB/c mice intravenously daily for 4 d, and peritoneal
mast cells were analyzed for Fc
RI by flow cytometry ~18 h
after the last injection. Administration of IgE, but not IgG2a,
resulted in a greater than twofold enhancement of peritoneal mast cell expression of Fc
RI (Fig. 7 A), whereas neither IgE nor IgG2a had any significant effect on mast cell expression of Fc
RII/III (data not shown).
Next, we assessed baseline levels of FcRI expression in
peritoneal mast cells of untreated IgE
/
versus IgE +/+
mice, and found that Fc
RI expression was reduced by
~83% in mast cells from IgE
/
mice as compared with
those from wild-type mice (Fig. 7 B). We also found that
administration of IgE (but not IgG2a) antibody resulted in
a marked increase in Fc
RI expression by IgE
/
mouse
peritoneal mast cells (compare Fig. 7, B and C), whereas
treatment with either antibody had no significant effect on
mast cell Fc
RII/III expression (data not shown). Moreover, there was a strong positive correlation, in the mice
shown in Fig. 7, A and C, between the log10 of the serum
IgE concentration at sacrifice and levels of mast cell Fc
RI
expression (R = 0.871, P <0.0001) (Fig. 8). And when
the peritoneal mast cells removed from the mice shown in
Fig. 7 C were tested for their ability to release 5-HT upon
antigen challenge in vitro, the response of mast cells from
mice that had been injected with IgE was markedly enhanced compared with that of the control cells from the
IgG2a-treated mice (Fig. 9).
Exposure to monomeric IgE in vitro markedly upregulated the ability of mature mouse peritoneal mast cells or
mouse BMCMCs or cloned mast cells to bind IgE. Two
separate lines of evidence indicate that this response largely,
if not entirely, reflected the increased surface expression of
FcRI. First, while mouse mast cells also express Fc
RII/
III (17, 18), which can bind IgE immune complexes (18),
virtually all of the binding of monomeric IgE to mouse
mast cells that is detectable under the conditions used in
our experiments reflects binding of the ligand to a single class of high affinity binding sites, i.e., Fc
RI (18). Second, we used anti-IgE to immunoprecipitate surface-bound IgE,
and associated IgE receptors, from lysates of BMCMCs that
had been first incubated with or without IgE at 5 µg/ml for
21 h and then exposed briefly to excess IgE just before recovery for flow cytometry and Western blot analysis. We
found that, in comparison to aliquots of the same mast cell
population that had been incubated without IgE, mast cells
that had been incubated for 21 h with IgE exhibited an
~14-fold increase in IgE binding by flow cytometry and an
~17-fold increase in IgE- and cell surface-associated Fc
RI
chain by Western blot analysis (Fig. 2 B). Thus, the IgEinduced increase in IgE binding that was detected in BMCMCs by flow cytometry was directly proportional to the
amount of Fc
RI
chain obtained from anti-IgE immunoprecipitates of the same cells.
The IgE-induced changes in levels of mast cell FcRI
surface expression that were detected in our experiments
reflect the operation of two processes, which can be distinguished by their sensitivity to inhibition by cycloheximide.
In confirmation of the findings of Furuichi et al. with RBL
cells (14), we found that the earliest component of the IgEdependent increase in BMCMC Fc
RI expression (which
occurred within 3 h of addition of IgE) was largely resistant
to inhibition by cycloheximide. By contrast, virtually all of
the subsequent sustained, and striking, upregulation of Fc
RI
expression in these cells was ablated by cycloheximide. The
most straightforward interpretation of these findings, which we favor, is that the initial, cycloheximide-insensitive, component of the response reflects IgE-dependent suppression
of the loss of preformed Fc
RI expressed on the cell surface
(14, 15), whereas the sustained, cycloheximide-sensitive
component is more dependent on protein synthesis, e.g., of
new receptors.
To address the in vivo relevance of our findings, we performed experiments in normal mice and mice that had been
rendered genetically IgE deficient. IgE /
mice expressed baseline levels of Fc
RI that were 83% reduced
compared with those of normal (IgE +/+) mice. Furthermore, both IgE
/
and normal mice exhibited significant
increases in the levels of peritoneal mast cell Fc
RI expression after i.v. treatment with IgE. These findings demonstrate that IgE is a major regulator of mouse mast cell
Fc
RI expression in vivo. Indeed, analysis of data from the
IgE-treated IgE
/
mice and the IgE +/+ mice that had
been treated with IgE, IgG2a, or PBS revealed a striking
positive correlation between the log10 of the serum IgE concentration at sacrifice and the log10 of the molecules of equivalent soluble fluorochrome (MESF) values for Fc
RI expression by peritoneal mast cells from the same mice (R = 0.871, P <0.0001). Thus, our findings in mice directly support the general form of the hypothesis that was originally proposed by Malveaux et al. with respect to human basophils
(13), namely, that circulating levels of IgE can influence
levels of effector cell surface expression of Fc
RI.
Nevertheless, mast cells from IgE /
mice exhibited
detectable, albeit greatly reduced, levels of Fc
RI on their
surface (Fig. 7 C), indicating that factors other than IgE
must also contribute to the regulation of mouse mast cell
Fc
RI expression. IL-4 has multiple effects that augment
IgE-dependent immune responses in mice; it is required for
normal IgE responses (30, 31) and can also promote the expansion of certain mouse mast cell populations (32, 33).
Moreover, peritoneal mast cells of IL-4
/
mice exhibit
fewer Fc
RI than do those of IL-4 +/+ mice (34). IL-4 can also upregulate levels of mRNA for the Fc
RI
chain
in human eosinophils (35). We found that recombinant
mouse IL-4 (at 10 ng/ml) slightly enhanced peritoneal mast
cell Fc
RI expression over that induced by IgE alone, but
had little or no effect in the absence of IgE (data not
shown). In light of this finding, one may speculate that the
reduction in mast cell Fc
RI expression in IL-4
/
mice
might reflect, at least in part, the low levels of IgE in these
animals (31, 34).
One of the most significant aspects of our study is the
observation that changes in the concentration of IgE antibody not only can regulate the level of cell surface expression
of FcRI on mouse mast cells, but that this IgE-dependent
upregulation of mast cell Fc
RI expression is functionally
significant. This result was, in some respects, unexpected.
In their studies of human basophils derived from patients
with different concentrations of circulating IgE, Conroy et al.
(11) detected no significant differences in the histamine release responses induced by different concentrations of antiIgE in basophils from nonatopic donors, which expressed
as few as 3,900 IgE molecules/basophil, or allergic donors,
which expressed up to 500,000 IgE molecules/basophil. Moreover, basophils from donors whose cells expressed approximately the same amounts of cell-bound IgE varied
greatly in their sensitivity to anti-IgE-induced histamine
release, perhaps reflecting a wide spectrum of intrinsic releasability of human basophils derived from different individuals (11).
By contrast, we found that IgE concentrations can regulate the expression of FcRI by mouse mast cells within a
range that has significant consequences with respect to IgEdependent effector cell function. Compared with mast cells
that had been incubated with levels of monomeric IgE similar to those present in the sera of some normal mice (i.e.,
0.005 µg/ml), mast cells that had been incubated with concentrations of IgE in the range observed in the sera of parasite-infected mice (i.e., 5.0 µg/ml) exhibited significant
mediator release at lower concentrations of antigen and also
gave significantly greater release of mediators at given higher concentrations of antigen. Thus, IgE-dependent upregulation of mast cell Fc
RI expression significantly enhanced both the sensitivity and the intensity of the secretory response of the cells to antigen challenge.
This effect was especially remarkable with respect to cytokine production. Thus, in comparison with cells that had been incubated for 4 d with IgE at 0.005 µg/ml, BMCMCs that had been incubated for 4 d with IgE at 5.0 µg/ml exhibited ~60% greater maximal release of the preformed mediator, serotonin, but ~6-fold greater release of the cytokine IL-6 (Fig. 4, B and C). The effect of IgE incubation on the ability of BMCMCs to produce IL-4 was even more striking. Substantial IL-4 production in response to antigen challenge was detected in BMCMCs that had been incubated for 4 d with IgE at 5.0 µg/ml, but was not detectable at all in cells that had been incubated for 4 d before antigen challenge either without IgE or with IgE at 0.005 µg/ml. The ELISA assay used for our studies has a lower limit of detection of <5 pg/ml of IL-4. It is possible (and perhaps likely) that different conditions of passive sensitization before antigen challenge, or the use of more sensitive indices of IL-4 production, may have permitted us to detect IgEdependent IL-4 production by BMCMCs that had not been incubated with high concentrations of IgE for 4 d before further passive sensitization and antigen challenge (7, 36, 37). However, under the conditions employed in our experiments, incubation of BMCMCs with IgE at 5.0 µg/ml for 4 d clearly markedly enhanced the ability of these cells to release IL-4 in response to challenge with IgE and specific antigen.
Taken together, our observations identify a novel and
potentially important mechanism for enhancing both the
sensitivity and intensity of IgE-dependent immune responses. Indeed, because IL-4 can markedly enhance the
generation of IgE responses in mice (30, 31), it is even possible that IgE-dependent upregulation of FcRI expression
by mouse mast cells, and subsequent enhancement of IgE-
and antigen-dependent mast cell IL-4 production, may constitute a previously unrecognized positive feedback loop in
the expression of IgE-dependent immune responses.
While the regulation of IgE production is complex, potentially involving multiple genetic and environmental factors, serum levels of IgE typically are greatly increased during parasite infections and are also elevated in most patients
with allergic diseases (38). Our findings suggest that any
mechanism that results in the substantial elevation of IgE
levels may also result in significantly enhanced IgE-dependent effector cell function. Furthermore, no matter what
may be the precise minimum number of cross-linked FcRI
that are required to trigger mast cell or basophil mediator
release (43, 44), cells that express increased numbers of Fc
RI
on their surface can be sensitized adequately with larger
numbers of different IgE species of distinct antigen specificities. Thus, increased expression of Fc
RI by mast cells would
also permit each cell to be simultaneously sensitized to respond to a larger number of different antigens.
In the context of acquired immune responses to parasites,
IgE-dependent enhancement of mast cell FcRI expression
would be expected to benefit the host. Unfortunately, the
same mechanisms might also increase the severity of allergic
diseases. Our preliminary experiments indicate that IgE can
also increase surface expression of Fc
RI on in vitro-derived
human mast cells (45). Accordingly, it will be of great interest to assess to what extent IgE-dependent upregulation
of Fc
RI expression, as here identified in mouse mast cells,
also occurs in human Fc
RI+ cells, and, if so, to determine
whether the ability of IgE to regulate Fc
RI can be exploited
therapeutically in the management of allergic disorders.
Address correspondence to Stephen J. Galli, M.D., Division of Experimental Pathology, Department of Pathology, RN-227, Beth Israel Deaconess Medical Center-East, Boston, Massachusetts 02215.
Received for publication 13 November 1996
Note added in proof: While this manuscript was in preparation, an in vitro study of mouse BMCMCs was published which contains results similar to some of the findings reported herein (Hsu, C., and D. MacGlashan, Jr. 1996. IgE antibody up-regulates high affinity IgE binding on murine bone marrow derived mast cells. Immunol. Lett. 52:129-134).We thank L. Fox, S. Fish, and H.-Y. Park for technical assistance, F.-T. Liu and D.H. Katz for H-1-DNP-26 hybridoma cells, K.E. Langley and AMGEN Inc. for rrSCF164, D.H. Conrad for B3B4 antibody, and
J.J. Costa, M. Tsai, and B.K. Wershil for a critical reading of the manuscript.
This work was supported by National Institutes of Health grants AI/CA-23990 and CA/AI-72074 (S.J. Galli), GM-53950 (J.-P. Kinet), K08 AI-01253 (H.C. Oettgen) and AI-26150 (I.M. Katona), and Uniformed Services University of the Health Sciences Protocol RO 86AB (to I.M. Katona).
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