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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1633-1643
In Vitro Regulation of Fc RI Expression on Human Basophils by
IgE Antibody
By
Donald MacGlashan Jr,
Jane McKenzie-White,
Kristine Chichester,
Bruce S. Bochner,
Frances M. Davis,
John T. Schroeder, and
Lawrence
M. Lichtenstein
From the Johns Hopkins Asthma and Allergy Center, Baltimore, MD; and
Tanox Biosystems, Inc, Houston, TX.
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ABSTRACT |
In vivo studies suggested the possibility of an IgE-dependent
regulation of high-affinity (Fc RI) IgE receptor expression on
basophils. The current studies extend these observations to in vitro
cultures of human basophils. Incubation of basophils for 3 to 4 weeks
resulted in a slow dissociation of IgE antibody, during which time
FcRI expression decreased, as measured by flow cytometry using the
anti-FcRI monoclonal antibody, 22E7, or by measuring FcRI
mass by Western blotting of whole-cell lysates. Culture of basophils
with IgE resulted in upregulation of FcRI expression by both flow
cytometry and Western blotting of whole-cell lysates. Upregulation
followed a linear time course during 2 weeks of culture. The relative
increase in FcRI density depended on the starting density; with
starting densities of FcRI of 10,000 to 170,000 per basophil, the
upregulation varied 20- to 1.1-fold, respectively. Upregulation
occurred in high-purity basophils, was not influenced by IgG at
concentrations up to 1 mg/mL, and was inhibited by dimeric IgE.
Heat-inactivated IgE was less effective and the monoclonal antibody
CGP51901 that prevents IgE binding to FcRI blocked the ability of
IgE to induce upregulation. The dose-response curve for IgE-induced
upregulation had an effective concentration50 of 230 ng/mL.
Although the receptor through which IgE induces this upregulation is
not yet known, several characteristics suggest that the upregulation is
mediated by IgE interacting through FcRI itself.
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INTRODUCTION |
THE HIGH-AFFINITY receptor for IgE,
Fc RI, is a critical component in the chain of events that lead to
type I hypersensitivity reactions, as this receptor allows mast cells
and basophils (as well as other recently identified cell types) to bind
IgE and therefore respond to environmental antigens. Consequently,
varied levels of expression of this receptor probably have an impact on
the severity of hypersensitivity reactions. It has not been clear how
expression of this receptor is regulated. However, in the late 1970s,
it was noted that there was an excellent correlation between the
density of FcRI on circulating basophils (measured as total IgE
density after saturation of the cells with IgE) and the serum IgE
titer.1 At that time, two hypotheses were advanced to
explain the correlation, one of which stated that IgE itself regulated
FcRI expression, while the other stated that there was an underlying
linkage that determined the relative expression of IgE and FcRI.
Overnight culturing of basophils with or without IgE antibody did not
show any change in FcRI densities. Because longer cultures were not
possible at that time, the question was left unresolved.
More recent experiments noted that unoccupied receptor densities were
remarkably constant across the entire 100-fold range of FcRI
densities observed on basophils from a wide range of donors.2 This result suggested that the cell acted to
maintain a low level of unoccupied receptors and, taken together with
the studies by Malveaux et al,1 further implied that IgE
levels determined the relative expression of FcRI. This was an
indirect argument and a more direct experimental protocol was necessary to support the belief that IgE itself regulated FcRI expression. Most recently, an in vivo test of this hypothesis was made possible by
monitoring basophil FcRI expression in patients receiving humanized
monoclonal anti-IgE antibody.3 This antibody resulted in
100-fold reductions in circulating free IgE concentrations and a 15- to
50-fold reduction in the expression of FcRI followed within weeks,
providing further support for the possibility that free IgE levels
determine the expression of FcRI on basophils. However, there were
other explanations for the effect observed in vivo and it remained
necessary to test the hypothesis using more controlled in vitro
studies. Recent studies using mouse bone marrow mast
cells4,5 or basophils,6 in vitro and in vivo, indicate that IgE regulates FcRI expression in this species. Human
basophils can now be cultured reasonably well for relatively long
periods in the presence of interleukin-3 (IL-3).7 Thus, we
examined the expression of FcRI on basophils cultured in the
presence or absence of IgE for extended periods. These studies demonstrate that IgE itself does appear to upregulate the expression of
FcRI on human basophils.
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MATERIALS AND METHODS |
Buffers.
Piperazine-N,N-bis-2-ethanesulfonic acid (PIPES; Sigma Chemical, St
Louis, MO) was used as stock buffer; 25 mmol/L PIPES containing 110 mmol/L NaCl, 5 mmol/L KCl, and 40 mmol/L NaOH was adjusted to pH 7.3 and stored at 10 times the above concentration. PAG consisted of PIPES
(1X) containing 0.003% human serum albumin (HSA; Miles Laboratories,
Elkhart, IN) and 0.1% glucose; PAGCM was PAG with 1.0 mmol/L
CaCl2 and 1.0 mmol/L MgCl2; PAG-EDTA was PAG
with 1.0 mmol/L EDTA. Acetate elution buffer contained 0.05 mmol/L
sodium acetate, 0.085 mol/L NaCl, 10 mmol/L EDTA and 0.03% HSA at pH
3.7.2 Borate buffered saline was 0.01 mol/L borate and 0.14 mol/L, NaCl at pH 8.4.
Reagents.
Polyclonal goat antihuman IgE was prepared as described
previously8; the antibody used for these studies
represented the IgG fraction of goat serum prepared by DE-52
chromatography. Penicillin (benzylpenicilloyl [BPO])-specific IgE was
partially purified from the serum of a penicillin-allergic patient by
affinity methods previously described in detail.9 It is a
combination of both IgG and IgE specific for penicillin, and more than
95% of the IgE is specific for the penicillin hapten. BPO(11)-HSA was
prepared as described previously.9 Purified
IgE-PS myeloma was a gift from Dr T. Ishizaka.10 Anti-nitrophenyl (NP) IgE was obtained from Dr
Robert Hamilton (Johns Hopkins) and anti-gp120 chimeric IgE obtained
from Tanox Biosystems (Houston, TX) was prepared by methods previously
described.11 CGP51901 was obtained from Tanox Biosystems
and its preparation and properties have been previously
described.12 For most of the experiments the IL-3 used for
culture was a gift from Dr Stephen Gillis, formerly of Immunex
(Seattle, WA). Later experiments also used IL-3 from Biosource (Camarillo, CA).
PS myeloma was heat-inactivated by treating a 10-mg/mL solution in
borate-buffered saline to 56°C for 90 minutes (previous studies have
used 30 minutes of heat activation, but this was found to be
insufficient). The extent of heat inactivation was tested by assessing
the ability of treated or untreated IgE antibody to inhibit histamine
release from basophils challenged with covalent dimers of IgE antibody.
Dimeric IgE-induced release requires challenge in the presence of
deuterium oxide to enhance secretion13 and release is
dependent on the presence of unoccupied receptors. Basophils were
preincubated with concentrations of IgE antibody (±heat treatment)
ranging from 10 ng/mL to 100 µg/mL for 30 minutes and then challenged
with 500 ng/mL dimeric IgE (a gift from Dr Henry Metzger) without
further washing. Histamine release was measured by automated
spectrofluorometry as described previously.
Cell preparation.
Two types of basophil preparations were used. Most of the studies used
cells obtained from leukapheresis and were prepared as previously
described.14 Basophil purities in these preparations ranged
from 15% to 95%, with a median of 33%. In some experiments, cells
were isolated from peripheral blood using the double Percoll method.15 The blood was diluted with EDTA-saline and
centrifuged at 500g for 15 minutes to obtain a buffy coat. The
buffy-coat cells were diluted in saline and layered onto a two-step
Percoll gradient: 1.065 g/mL/1.079 g/mL as described
previously.16 After centrifugation at 450g for 15 minutes, the interface between the 1.065 Percoll/plasma upper layer and
the 1.079 lower Percoll layer was harvested and washed as described
earlier (basophil purities, 8% to 45%). Which cell preparations were
used will be noted in the text.
Cell culture.
Enriched basophil preparations were cultured in Iscove's modified
Dulbecco's medium (IMDM; Life Technologies, Gaithersburg, MD) containing 2% fetal calf serum (FCS), 40 µg/mL gentamycin, and
10 ng/mL IL-3. The total cell density was 2 × 106/mL and
culturing was performed in 96-, 24-, or six-well tissue culture-treated
plates (Costar, Cornell, NY).
Unoccupied receptor densities.
In a few experiments, the cells were sensitized with BPO-specific IgE
before its elution to determine unoccupied receptor densities. Briefly,
unoccupied receptors were saturated with BPO-specific IgE, the cells
counted, the IgE eluted with a acetate elution buffer, and the IgE
quantified in a BPO-specific IgE radio allergo sorbant test
(RAST).2 The combination of cell counts and
the amount of IgE eluted allows a calculation of the unoccupied
receptor density.2 A similar procedure is used to determine
endogenous IgE density except that a total IgE radio immuno sorbant
test (RIST) is used to measure the eluted IgE antibody.2
From the rate that unoccupied receptors were loaded during
sensitization, the on-rate constant (kon) was estimated.
Short preincubations with high concentrations of IgE or longer
incubations with lower concentrations of IgE were used to estimate this
value. A similar protocol was used to determine the degree of FcRI
saturation following various incubations with PS myeloma IgE. Basophils
that were incubated with PS myeloma IgE were then tested for the
presence of remaining unoccupied receptors by loading the remaining
receptors with BPO-specific IgE and eluting as described earlier.
Basophils that had not been incubated with IgE myeloma provided a
measure of the starting unoccupied receptor densities.
Western blotting.
High-speed cell pellets ( 14,000g for 5 to 10 seconds) were
resuspended at 3 × 107 cells/mL in lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 5 mmol/L EDTA, 10 mmol/L EGTA, 5 mmol/L
dithiothreitol, 1% nonidet P-40, 1 mmol/L phenylmethylsulfonyl
fluoride [PMSF], 20 µg/mL leupeptin, 100 µg/mL aprotinine, and 10 mmol/L benzamidine). After 20 seconds of vortexing, the cell lysates
were kept on ice for 20 minutes, and microfuged for 15 minutes at
4°C. Supernatant was collected as a protein extract that contained
lysed cell components without nuclei.17 Previous studies
have established that lysing basophils or contaminating cells with
these buffers generates equivalent protein levels. Protein levels in
the extracts from different days may have been different, as this was
not measured, but normalizing with respect to cell number was the
information desired, rather than protein levels, which might change
during culture in IL-3. Extracts that contained equal basophil cell
numbers (3 × 105 cell equivalents per lane) were
diluted with an equal volume of 2× loading buffer (0.125 mol/L
Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate [SDS], 0.005%
bromophenol blue, 20% glycerol; NOVEX, San Diego, CA) containing
0.05%  -mercaptoethanol, and subjected to 4% to 20% Tris glycine
density gradient gel electrophoresis (NOVEX). Gels were then
transferred to pure nitrocellulose membranes (Schleicher & Schuell,
Keene, NH) by Trans Blot (NOVEX).
Electrophoresis and transfers were performed according to the
manufacturer's recommendations. After transfer, membranes were immersed in Tris-buffered saline with Tween 20 (TBST) that
contained 5% nonfat dry skim milk (Carnation, Los
Angeles, CA) overnight to block nonspecific binding. Membranes were
then washed three times for 5 minutes with TBST. Immunoreactive
proteins were detected using the 22E7 antibody diluted to 5 µg/mL in
TBST containing 1% skim milk. After a 4-hour incubation, membranes
were washed with TBST and incubated with peroxidase-labeled sheep
antimouse Ig antibody (Amersham, Arlington Heights, IL) for 1 hour.
After five 10-minute washes, membrane-bound antimouse Ig antibody was visualized by enhanced chemiluminescence (ECL) Western blotting detection reagents (Pierce, Rockford, IL), and Hyper-ECL
luminescence detection film (Amersham). The ECL films were
converted to digital format with a UVP (Upland, CA) digital camera and
the images analyzed with NIH Image (Wayne
Rasband).14 Pilot studies using human basophils (99.8%),
mast cells, lymphocytes and monocytes demonstrated a detectable band
only in the basophil preparations, an extremely weak band in the mast
cells, which generally have low receptor densities,18 and
no bands for the lymphocytes or monocytes.
Flow cytometry.
A flow-cytometric technique incorporating light-scatter characteristics
was used to quantify cell-surface IgE and FcRI chain expression
on basophils as described.19 Cell-surface IgE was detected
using a monoclonal antihuman IgE (TES-19 or E10-10-3; Tanox
Biosystems). Cell-surface expression of FcRI chain was detected
using a mouse IgG1 antihuman FcRI chain monoclonal antibody
(22E7; generously provided by J. Kochan, Roche
Pharmaceuticals, Nutley, NJ20) and was compared to
labeling with an identical concentration of irrelevant mouse IgG1
(Coulter, Hialeah, FL). The 22E7 antibody has been shown to recognize
an epitope that is unaffected by FcRI occupancy.20 In
several experiments, the monoclonal antibody 15A5 was also used. This
antibody binds only to unoccupied FcRI.20 Aliquots of
cells were labeled in phosphate-buffered saline that contained 0.2%
HSA with 1 mg/mL human IgG to minimize nonspecific binding to
Fc R.19 Each of the monoclonal antibodies was used at
concentrations predetermined to be optimal for labeling. Binding of
monoclonal antibodies was detected using saturating concentrations of
R-phycoerythrin (PE) conjugated polyclonal goat antimouse IgG (Tago,
Burlingame, CA). An EPICS Profile flow cytometer (Coulter) was used to
analyze fluorescent signals after excitation at 488 nm. Bitmap gates,
intermediate between the forward- and side-scatter characteristics of
lymphocytes and monocytes, were used to select for a population of
cells that were predominantly basophils. Since the cells were already
enriched in basophils, these bitmaps can select a population of cells
that is generally greater than 80% basophils, with the primary
contaminants being lymphocytes. Data are expressed as the mean
fluorescence in labeled cells minus the mean fluorescence of IgG1
controls. Day-to-day variability in the sensitivity of the flow
cytometer was corrected by noting or adjusting the photomultiplier tube
voltage to generate the same signal for a set of standard calibration
beads (Immunochek; Coulter).
The cells used to study basophil survival and apoptosis were obtained
by venipuncture and two-step Percoll gradient enrichent. When annexin-V
labeling was used, the cell cultures were harvested, centrifuged once,
and resuspended in buffer that contained anti-IgE antibody, TES-19.
Following a 10-minute incubation, the cells were centrifuged once and
resuspended for annexin-V binding according to the manufacturer's
directions (ApoAlert; Clonetech, Palo Alto, CA), with the exception
that antimouse PE was included in the buffer. Following
an additional 10-minute incubation, the cells were not subjected to
further washing, but directly examined by flow cytometry. Forward- and
side-scatter bitmaps were used to select the approximate population
containing basophils, and these cells were further gated for annexin-V
binding by selecting those that were stained with anti-IgE antibody.
The flow-cytometric measurements were calibrated by examining the
fluorescence staining of six donors' basophils that spanned a moderate
range of staining intensities (7 to 120 fluorescent units or 8,000 to
140,000 FcRI per basophil) and simultaneously assessing receptor or
IgE density by the acetate elution method described earlier. 22E7
staining (ordinate) compared with total FcRI density by acetate
elution (after sensitizing with PS myeloma IgE as described earlier)
was linear with a slope of 0.00084 (ie, a fluorescence measurement of
100 represents approximately 120,000 receptors) with R = .963. Likewise, total IgE density versus fluorescent staining with E10-10-3
anti-IgE antibody (ordinate) was linear with a slope of 0.00145 and
R = .992.
Statistics.
Student's t-test was used for most statistical comparisons,
while others were made with a nonparametric Wilcoxon signed-rank statistic or analysis of variance (ANOVA). If errors or error bars are
shown, they represent the standard error of the mean unless otherwise
indicated.
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RESULTS |
Downregulation of FcRI.
These studies required long incubations of basophils, so the
survivability of basophils in these relatively long cultures (mature
basophils have not been observed to proliferate) was assessed. Figure
1 shows data derived from basophils
isolated from whole blood and enriched by two-step Percoll gradients.
Substantial variability was observed among basophil preparations during
the first 2 weeks, although there was a consistent 75% loss by 3 weeks. These cells were also examined for apoptotic changes as assessed by the binding of annexin V. In these experiments, cells carefully harvested from culture (see Methods) were colabeled with anti-IgE antibody/antimouse-PE and fluorescein isothiocyanate (FITC) annexin V. For flow cytometry, cells were gated according to both forward- and
side-scatter characteristics and labeling with anti-IgE antibody. It
was subsequently found that about the time a small fraction of the
basophils acquire a moderate labeling with FITC-annexin V, these same
basophils fall into a lower region of the forward-side-scatter profile.
This process occurred throughout the 3-week period, which indicates
that the relatively high concentration of 10 ng/mL of IL-3 was not able
to prevent basophils from entering apoptosis over this time frame.
Attempts to culture enriched basophils in 20% autologous serum did not
improve survival (data not shown). For the experiments that follow, the
forward-side-scatter gates were set to exclude the region most
associated with these apoptotic basophils.
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| Fig 1.
Loss of basophils during culture in IL-3 (n = 7,
except the last data point,  , where 2 experiments were extended to
25 days). Data are plotted as the fraction of the starting basophil
number, means ± SD.
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In the in vivo studies of anti-IgE treatment, the treatment led to a
marked decrease in FcRI expression. To examine whether the same
decrease would occur in vitro, basophils were cultured in the absence
of IgE in the media for 14 to 21 days and were tested for FcRI
expression using the FcRI-specific monoclonal antibody 22E7 and
for cell-surface IgE using a mouse monoclonal anti-IgE antibody, TES-19
or E10-10-3. The data shown in Fig 2 were
derived from basophils that were obtained by the double Percoll method
and represent partially enriched basophil preparations ranging from 5%
to 40% in purity. Figure 2 shows that basophil-bound IgE declined,
with the 50% reduction point occurring only after approximately 8 days. There was a lag in the decrease in FcRI so that the 50%
point was reached only after 13 days. Similar results have been
observed in preparations of basophils obtained from leukapheresis
packs, which range in purity from 15% to 95% (data not shown). While
FcRI expression decreased, the distributions remained unimodal
(one example from these four experiments, with measurements made on day
0 and day 17, is shown in Fig 2B and C, respectively).
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| Fig 2.
Downregulation of both IgE and FcRI during culture
in the absence of IgE in the culture medium (n = 4). Both
parameters were measured by flow cytometry, IgE with TES-19 anti-IgE
monoclonal antibody ( ) and FcRI () with monoclonal antibody
22E7. On average, the starting levels of expression for FcRI were
110 ± 25 flow fluorescence units, equivalent to 130,000 FcRI
per cell (see Materials and Methods). Data are expressed as a fraction of the day 0 level of expression. (B and C) Representative
flow-cytometric profiles for 1 of the experiments; (B) day 0 distribution (mean fluorescence, 63), and (C) day 17 distribution
(mean = 17) using 22E7 to detect FcRI.
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The slow rate of dissociation of IgE from the basophils in these
cultures was not predicted. Published accounts of the affinity of human
IgE for FcRI would predict 50% dissociation occurring in the first
12 to 48 hours.21-23 The inclusion of an anti-IgE antibody
to capture dissociating IgE should eliminate the various ways that IgE
may rebind. In a short series of experiments (Table 1), enriched basophils were cultured
without IgE and with or without CGP51901, a chimeric monoclonal
antibody that binds to IgE on an epitope hidden when IgE is bound to
the FcRI subunit. Therefore, this antibody does not bind to IgE
bound to FcRI, but does bind to dissociated IgE with an affinity
high enough12 to minimize dissociated IgE rebinding to
FcRI. Table 1 lists the results from three experiments in which
the loss of cell-surface IgE on basophils was monitored during culture
for several days. The test conditions were cultured with or without CGP
51901, which was tested at either 10 or 200 µg/mL. On theoretical
grounds,24,25 both concentrations of antibody should
adequately remove any dissociating IgE; however, the higher
concentration was included to provide a vast excess of antibody. These
data indicate that inclusion of CGP51901 had no effect on the rate of
IgE dissociation, which suggests that the observed rate is a true
reflection of the high affinity of the receptor for IgE under these
particular conditions.
In the first 10 days of culture, there is a significant loss in
cell-surface IgE expression that is not accompanied by as great a loss
in the receptor. The implication is that there should be a larger
number of unoccupied FcRI than found at the start of the culture.
This interpretation was first supported by examining the ratio of
fluorescence obtained by labeling with 22E7 versus TES-19 antibodies at
various times during culture. For most donors, the ratio of total
receptors to total IgE is close to 1.0 when freshly
isolated,2 so the ratio of fluorescent intensities following labeling with these two primary antibodies under our standard
conditions of flow cytometry is relatively constant, 0.64 ± 0.05 (22E7/TES-19) (n = 6 for the following comparisons). After 10 days of
culture, the ratio becomes 1.29 ± 0.14 (different from the starting
ratio, P < .01), which indicates significantly more
receptors than IgE (and therefore more unoccupied receptors). If these
day 10 cells were cultured with 10 µg/mL of IgE for 20 minutes or for
1 day, the ratio returned to 0.54 ± 0.12 or 0.69 ± 0.12, respectively. These indirect results were verified by examining the
cells at various times during the first 2 weeks of culture with the
monoclonal antibody 15A5, which binds only to unoccupied FcRI.
15A5 binding steadily increased and stabilized by 10 to 14 days at a
value 585% of preculture levels (n = 2, data not shown). A second
method used the acetate elution method of measuring unoccupied
receptors. This method counts unoccupied receptors by first filling the
receptors with a BPO-specific IgE, eluting the IgE, and measuring the
eluted IgE in a BPO-RAST. With cell counts available, the absolute
unoccupied receptor density can be calculated. In the one experiment
performed this way, unoccupied receptors started at 6,300 ± 900 and
increased to 26,000 ± 3,000 by day 11.
Modest IgE antibody-dependent FcRI downregulation and upregulation
has been observed in rat basophilic leukemia (RBL) cells.26 For the RBL cell, IgE appears to stabilize the
cell-surface expression of FcRI by preventing its endocytosis. We
therefore tested for the presence of FcRI in human basophils
following its downregulation in culture by lysing the cells for Western
blotting. Figure 3 shows the results for
21-day cultures. As found previously, the average expression of
FcRI as determined by flow cytometry was 30% to 40% of day 0 levels; the Western blotting data reflected a similar loss, not
statistically different than the data for flow cytometry.
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| Fig 3.
Changes in the cell-surface expression of FcRI as
measured by 22E7 binding or changes in total cell FcRI as
measured by Western blotting. (A) An example of the Western blot data
for cells at day 0 or day 21 after culture. The number of total cells is held constant and the basophil purity was found to be the same for
the 2 time points, averaging 41% ± 13% for the 5 experiments. (B)
Average data for flow-cytometric measurements or Western blotting in
terms of day 0 levels (n = 5) Western blot films were digitally imaged to determine optical densities of the bands. The difference in
relative change as measured by flow cytometry or Western blotting was
not statistically significant, while both were significantly less than
day 0 (P < .001).
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Upregulation of FcRI.
The remaining experiments focused on the upregulation of FcRI
expression. One implication of our recent in vivo studies is that
FcRI expression is markedly upregulated during the life span of a
basophil. Unpublished experiments in the early 1980s had shown that no
significant upregulation of FcRI occurred in a 24-hour culture. We
anticipated that upregulation induced by IgE would be more observable
if the starting receptor density was moderately low at the onset of a
culture with IgE antibody. Therefore, for many preliminary experiments
testing this hypothesis, the cells were first cultured for
approximately 21 days to downregulate FcRI. Figure
4 shows the kinetics of upregulation in the
presence (or absence) of 500 ng/mL of PS myeloma IgE in these cells
that had been first cultured for 21 days. The upregulation is
relatively linear for the 7-day period examined and averages an
increase of 2.6 ± 0.3-fold by 7 days. The flow-cytometric
distributions for FcRI expression remained unimodal throughout
the experiments (an example from one of these five experiments is shown
in Fig 4B and C for day 0 and day 7, respectively). Note that in the absence of IgE, FcRI expression continued to fall beyond the point labeled day 0, which is actually day 21 after the isolation of
the cells (for cells cultured in the absence of IgE, the day 1, 2, and
3 measurements were derived from only three of the five experiments).
In two of these experiments, the incubation in the presence of IgE was
performed for 2 weeks. However, no further upregulation beyond the
1-week values was found (data not shown), although it was noted that
only 5% to 10% of the basophils survived into the fifth week.
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| Fig 4.
Upregulation of FcRI expression on basophils during
a 7-day culture in the absence () or presence () of 500 ng/mL of
IgE (n = 5). Cells used for these experiments had already been
cultured for 21 days to downregulate FcRI. Average starting level
of expression was 37 ± 13, which translates to approximately 45,000 FcRI per basophil (see Materials and Methods). Data are expressed as the increase relative to the day 21 levels of expression (the start
of the upregulation phase of the cultures). For cells cultured in the
absence of IgE, measurements were made on days 1, 2, and 3 for only
three of five experiments ( ); day seven measurements were made for all five experiments. (B and C) Representative
flow-cytometric profiles for 1 of the experiments; (B) starting
distribution (mean fluorescence, 27), (C) distribution (mean, 86) after
7 days of culture with IgE.
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The magnitude of the upregulation was not as marked as might be
anticipated. Based on in vivo studies, receptor densities could cover a
50-fold range. It seemed likely that the higher the starting density,
the smaller the subsequent fold-increase (see below and Discussion).
Figure 5 shows results compatible with this
perspective. The data in Fig 5 are derived from a number of sources.
Two types of preparations were studied using cells from leukapheresis
packs: cells obtained from leukocyte packs that had been first
downregulated by 21-day culture or leukapheresis pack cells in which
IgE was added to the cultures immediately after isolation. Likewise,
cells obtained by standard venipuncture techniques and double Percoll
isolation were also used immediately for upregulation or upregulated
after a 21-day culture period. The upregulation was clearly inversely
related to the starting level of FcRI expression. With starting
levels below 20 fluorescence units (which we have calibrated to be
equivalent to 20 to 25,000 receptors per cell, see Materials and
Methods), there is greater than fivefold upregulation over 7 days. In
one extreme case of a very low starting density, a nearly 20-fold
increase was observed. In six experiments in which the cells were
immediately placed into culture for upregulation, a 2-week measurement
was also made. In all cases (starting levels of 21 to 98 fluorescence
units), the 2-week levels of FcRI expression were higher than the
1-week levels (1.6-fold to 2.6-fold), and in two experiments in which the starting levels averaged a moderately low 22 fluorescence units,
the 1- and 2-week increases averaged 2.6- to 4.8-fold, respectively. It
should also be noted that the IgE concentration in these cultures did
not appreciably decrease during culture. IgE concentrations after 1 week of culture were 0.96 ± 0.05 of the starting concentration, as
determined in a RIST. Furthermore, the ability of this 1-week-old IgE
to sensitize basophils remained intact. This was tested by culturing a
large number of basophils in the absence of IgE, as well as by
culturing a smaller number of the same cells in the presence of IgE.
After 1 week, the culture supernatant that contained IgE was recovered
and used to make serial dilutions of IgE, which were used to sensitize
the cells that had not been cultured with IgE. The dose-response curve
for sensitization of the later cells with the "old" IgE was
examined by testing the cells after overnight culture for an ability to further bind BPO-specific IgE. The titration curves of old versus fresh
IgE at the same concentrations were the same. Taken together, these
data indicate that there was no functional loss of the IgE that is
present in 1-week cultures.
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| Fig 5.
Upregulation of FcRI of different types of basophil
preparations under the influence of 500 ng/mL of PS myeloma IgE during a 7-day culture. The four symbols represent different methods to obtain
basophils; (*) basophils obtained from leukapheresis packs and
incubated for 21 days before addition of IgE, () basophils obtained
from leukapheresis packs but cultured for 7 days without the
downregulation step of 21-day culture, () basophils obtained from
standard venipuncture and double Percoll gradient separation and
incubated for 21 days before addition of IgE, ( ) basophils obtained
from standard venipuncture and double Percoll gradient separation but
cultured for 7 days without the downregulation step of a 21-day
culture. Each symbol represents the results from a single donor. The
starting receptor density (x-axis) is the 22E7 fluorescence by flow
cytometry and the fractional upregulation (y-axis) is the ratio of day
7 fluorescence to day 0 fluorescence.
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With the more typically modest upregulation, it appeared possible that
upregulation only involved the cell-surface expression of cryptic
receptors.26 In such a situation, lysis of the cells and
analysis by Western blots should show no change in the total cellular
mass of FcRI during the period of upregulation. However, this was
not the case. As shown in Fig 6, following
a 1-week culture in the presence of IgE, there was a statistically
significant increase (P = .01) in the mass of FcRI:
approximately twofold in the experiments shown. Likewise, the
cell-surface expression increased approximately twofold in these
particular experiments.
View larger version (30K):
[in this window]
[in a new window]
| Fig 6.
Upregulation of FcRI as determined by either flow
cytometry or Western blotting of lysed cells (n = 6). (A) Example
of Western blot data for cells at day 0 or day 7 after culture in the
presence of 500 ng/mL of PS myeloma IgE; day 0 is the day that IgE was added to the cultures, which was generally after a 21-day culture to
downregulate the receptors (basophils were obtained from leukapheresis packs). The number of total cells was held constant and the basophil purity was found to be the same for the two time points, averaging 45% ± 5% for the six experiments. (B) Average data for flow-cytometric measurements or Western blotting in terms of day 0 levels (n = 6) Western blot films were digitally imaged to determine the optical densities of the bands. The difference in the relative change as
measured by flow cytometry or Western blotting was not statistically significant, but both measurements for day 7 were statistically different than day 0 (P = .022 and P = .010,
respectively). Average starting fluorescence of the basophils was 52 ± 15 on day 0, corresponding to 60,000 receptors per basophil (see
Materials and Methods).
|
|
The ability of IgE to upregulate FcRI was not limited to PS
myeloma; upregulation occurred with other IgE antibodies, an anti-NP
IgE and an anti-gp120 IgE. IgG at similar concentrations or up to 1 mg/mL caused no upregulation of FcRI nor did it inhibit the
upregulation by IgE (Table 2). It is
possible that aggregates of IgE are required to observe upregulation;
however, we have found that covalently crosslinked dimers of IgE
antibody did not induce upregulation of FcRI (however, these dimers
induced histamine release when basophils were challenged in the
presence of deuterium oxide, as previously found,27 see
Materials and Methods). A combination of dimeric IgE and monomeric IgE
resulted in less upregulation than monomeric IgE alone (Table 2).
Treating the cells with a crosslinking goat polyclonal anti-IgE
antibody (0.1 µg/mL) caused downregulation of the receptor. In two
experiments, basophils were cultured in the presence or absence of
anti-IgE antibody for 3 days. FcRI expression remained constant for
cells cultured without anti-IgE, while FcRI decreased to 62% and
42% of day 0 levels for the two experiments for cells cultured with anti-IgE antibody. Last, in two experiments, basophils of 90% and 97%
purity demonstrated upregulation that was no different that from cells
of lower purity with similar starting levels of FcRI expression
(Table 2).
Figure 7 shows the concentration dependence
of this upregulation. Basophils were cultured for 7 days in the
presence of various concentrations of IgE and again assayed for the
expression of FcRI. Surprisingly, maximal upregulation appeared
to require IgE concentrations greater than 5 µg/mL; the difference
between 500 and 5,000 ng/mL is statistically significant. In two
experiments, we tested 50 µg/mL of IgE and found small decreases
(12%) in the level of upregulation compared with that observed for 5 µg/mL. Based on the data for these six experiments and the apparent
saturation at approximately 5 µg/mL, the effective
concentration50(EC50) for upregulation averaged
230 ng/mL.
View larger version (12K):
[in this window]
[in a new window]
| Fig 7.
Concentration dependence of IgE-induced upregulation of
FcRI in basophils cultured for 7 days (n = 6). The abscissa
is expressed as the ratio of 22E7 fluorescence for cells incubated with
IgE at the various concentrations to cells not incubated with IgE. () Data for PS myeloma; () data for heat-inactivated PS myeloma. The average starting fluorescence of the basophils was 25 ± 4, corresponding to 30,000 receptors per basophil (see Materials and
Methods). Basophils were obtained from leukapheresis packs and first
downregulated by a 21-day culture.
|
|
Heat-inactivated PS myeloma was examined for its ability to upregulate
FcRI in 1-week cultures. The heat-inactivated IgE was tested in
the dose-response curve experiments shown in Fig 7. In these
experiments, heat-inactivated IgE was 20-fold less potent than the
untreated IgE. It should be noted that if the heat-inactivated IgE was
used to inhibit the ability of dimeric IgE to induce histamine release
in basophils (see Materials and Methods), it was found to be
approximately 100-fold less potent than untreated IgE.
In the same context, the importance of the FcRI binding domain of
IgE (and the region surrounding this part of IgE) was examined by
testing the effects of CGP51901 on upregulation. As noted earlier,
CGP51901 binds to this epitope of IgE, inhibiting its ability to bind
to FcRI and CD23. It is not known to bind to IgE already bound to
FcRI. Basophil preparations were treated with or without IgE and
with or without CGP51901 at a 20-fold excess concentration (10 µg/mL). Table 3 shows that, by itself, CGP51901 had no effect on the downregulation of FcRI, but did inhibit the IgE-mediated upregulation (ANOVA, P = .0001, with P < .001 for columns 2, 3, or 5 different from column 4, the treatment with IgE alone). Upregulation was modest in these
experiments, because fresh cells were studied with moderately high
starting FcRI expression.
| |
DISCUSSION |
These studies show a change in the expression of FcRI on human
basophils when IgE antibody is excluded or included in the media used
for culturing. Both downregulation and upregulation of the receptor
under these culture conditions is relatively slow and appear to result
from the loss or gain of FcRI mass. Both of these observations
occur with relatively high-purity basophils (up to 97%). If a cell
other than the basophil is involved in the upregulation process, very
low numbers are required. IgG antibody is unable to effect an increase
nor does it inhibit the upregulation by IgE antibody. Therefore, it
seems unlikely that there is interaction of IgE through the IgG
receptor on basophils (CD32, FcRII). Aggregation of IgE does not
appear to be necessary for upregulation, as polyclonal anti-IgE induces
modest downregulation over a period of days and dimeric IgE does not
appear to cause either upregulation or downregulation. Further, dimeric
IgE mixed with monomeric IgE results in less upregulation than
monomeric IgE alone. Taken together, these data suggest that monomeric
IgE interacts with a receptor on basophils to induce upregulation of
FcRI. It is not clear why dimeric IgE does not induce some
upregulation, but there may be a balance of downregulation, as a
consequence of aggregation, with the upregulatory effects of monomeric
binding.
Ig-dependent expression of other Fc receptors is a common theme in the
immune system. In particular, IgE is known to modulate the expression
of CD23 (FcRII) on B cells.28,29 Likewise, IgA and IgG
have been found to regulate the expression of their respective
receptors.30-32 In the case of CD23, IgE appears to stabilize the cell-surface expression of FcRII by preventing its
proteolytic cleavage from the cell surface.29 Indeed, the lifetime of a CD23 molecule on the cell surface appears short if IgE is
not bound. Unlike the CD23/lymphocyte model, the model in RBL cells
appears to depend on endocytosis of FcRI when IgE is not bound
and it appears that FcRI is recycled so that there remains an
internal pool that is constantly cycling to the surface.26 The human basophil does not appear to strictly follow this model, since
the model would predict no loss in the mass of FcRI during the
loss of cell-surface expression, while in our experiments, the mass of
FcRI was lost during the period of downregulation. However, we
cannot yet distinguish between loss from the cell surface to the medium
and loss by endocytosis followed by degradation. In the RBL cell, the
upregulation is limited to the stabilized appearance of presynthesized
FcRI and this limits the level of upregulation to twofold to
threefold. These numbers are similar to those in a typical basophil
experiment and might also suggest that a similar mechanism plays a role
in human basophils. However, we can also observe an increase in the
mass of FcRI during upregulation, which did not occur in the RBL
cell studies.
Our in vivo studies reported downregulation of FcRI that averaged
26-fold, with a range from fivefold to 50-fold. The range of FcRI
receptor densities among the general population is approximately 100-fold, although those at the low end are relatively rare. Thus, one
might expect at least 10-fold changes in FcRI expression in these
culture experiments. The slow loss of basophils in these relatively
long cultures and the appearance of apoptotic basophils suggests that
the relatively modest levels of upregulation in the presence of
exogenous IgE may result from basophils cultured under suboptimal
conditions (or the loss of basophils reflecting their natural lifespan,
which has been estimated to be up to 2 weeks in vivo). Despite these
unfavorable conditions, the increase in FcRI in human basophils
was not limited to twofold to threefold. The starting density of
FcRI was the strong determinant of the extent of upregulation. In
the few cases in which the starting density was as low as we have
observed after 3 months of treatment with therapeutic anti-IgE
antibody,3 and the cells were cultured with IgE the day
that they were first obtained, larger increases in FcRI were
noted and the process appeared linear for at least 2 weeks. For similar
starting densities, both freshly isolated basophils and cells first
cultured for 21 days showed similar upregulation, which suggests this
effect was not limited to the select small percentage of basophils that
survived for 3 to 4 weeks. Taken together, the in vivo studies and the
current in vitro studies indicate a far more dynamic range of
regulation, which is probably not simple recycling. We have observed
eightfold upregulation in bone marrow-derived mast cells cultured with
IgE,4 and studies by Yamaguchi et
al5 and Lantz et al6 have found that
upregulation of FcRI on mouse mast cells can be greater than 10-fold
and is likely to result from the synthesis of new receptors.
From these studies, only a limited conclusion can be drawn regarding
the receptor involved in mediating the effects of IgE on FcRI
expression. There are a limited number of known choices; these include
FcRI, FcRII, CD32,33 or BP,34 and
future studies will address some of these possibilities further (until recently, IgE-dependent histamine-releasing factor would have been
included in this list, but it is not currently believed to bind
IgE35,36). With respect to IgE interacting with FcRI, the high concentration-dependence of the upregulation (Fig 7) is
difficult to interpret at this time. The interpretation of the data is
somewhat dependent on the Ka one chooses to
accept for the IgE/FcRI interaction. The literature value of
approximately 1 × 1010 translates to
half-occupancy at approximately 20 ng/mL.21-23,37 Provided
our IgE is fully intact and 100% is able to bind to receptor, this
value is less than one tenth of our noted EC50 value. This raised the possibility that the PS myeloma being used did not have the
expected affinity for FcRI. However, as noted earlier, other
preparations of IgE resulted in similar data, which suggests the
apparently lower affinity was not restricted to one IgE preparation. Although the apparent shift in the concentration dependence (for binding to FcRI) will need further study, it may reflect the nature
of the underlying mechanism for upregulation. Upregulation may be
dependent not on equilibrium conditions, which are unlikely to be
completely established in these cultures given the low rate of
association, but on the binding dynamics. With an association rate
constant ranging from 3 × 104 to 1 × 105
mol/L 1 · s1, low concentrations of
IgE may require many days to reach the equilibrium point so that if the
mechanism is dependent on the stability of an unoccupied versus
occupied receptor, this binding dynamic becomes important. The current
data do not support a role for CD23. In human lymphocytes, FcRII
affinities for IgE would suggest an EC50 more than 10 µg/mL,38 indicating that the EC50 of 230 ng/mL is a far lower concentration than expected if IgE were
interacting with FcRII. In B lymphocytes, dimeric IgE induced better
upregulation of CD23,28 which suggests that if CD23 is involved in upregulating FcRI in basophils, then dimeric IgE might
be a better signal. However, the opposite was true in these studies.
Finally, although flow cytometry is relatively insensitive and may not,
therefore, provide a proper assessment of low-density receptors,
flow-cytometric studies have not detected CD23 on human basophils. The
data concerning the effects of IgG do not support a role for CD32. On
balance, the data suggest that IgE exerts its influence through
FcRI itself. Future studies will need to verify this conclusion.
The kinetics of downregulation seemed appropriate for the rate that IgE
was found to dissociate from the basophils. However, the slow rate of
IgE dissociation was puzzling. The literature for normal human IgE
interacting with FcRI on normal human basophils or mast cells is
sparse. A variety of methodologies, some using assumptions invalidated
by the current studies, have produced dissociation rates that range
from 322 to 24 hours.1,39,40 On a theoretical
level, there should be no significant rebinding of IgE to FcRI
that would explain the slow dissociation in the human basophil cultures
described here.24,25 Nevertheless, any type of
reassociation, if it occurred, should have been eliminated by capturing
the IgE with CGP51901 anti-IgE antibody, preventing its rebinding,
without actually physically removing it from solution. A variety of
studies have examined the dissociation of IgE from FcRI on RBL cells
and this process may be biphasic.41 The two time constants
(T1/2) range 4 to 40 hours42-45 for the fast
component and 150 to 250 hours for a second component. This later
component, the cause of which is not known, is consistent with the long
dissociation rate found in our human basophil studies. The
downregulation observed in these long-term cultures is also unlikely to
result only from the slow progression of basophils into apoptosis,
since it was possible to hold receptor levels constant with an
appropriate concentration of IgE antibody. In addition, the in vivo
anti-IgE antibody treatment studies also suggest that the decrease in
FcRI expression accompanies lower free IgE titers.3
If IgE can signal through FcRI itself to induce its expression, this
represents a new role for this receptor. Future studies will be
necessary to determine whether expression of this receptor and its
regulation by IgE are dependent on changes in receptor synthesis
and/or catabolism.
| |
FOOTNOTES |
Submitted July 1, 1997;
accepted October 14, 1997.
Supported in part by National Institutes of Health Grants No. AI20253
and AI07290.
Address reprint requests to Donald MacGlashan, Jr, PhD,
Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Cir, Baltimore, MD 21224.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract