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RI
Surface Densities and Mediator Release by Anti-IgE-Infusions Is Reversible In Vitro and In Vivo1
*
Department of Medicine, Division of Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore MD 21224; and
Genentech, South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI
surface density, and polyclonal anti-IgE and Ag-induced basophil
histamine release responses. We hypothesized that these effects would
be reversed in vivo by discontinuation of infusions and in vitro by
exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46
wk. Blood samples taken 0–52 wk after rhumAb-E25 were analyzed for
serum IgE and basophil expression of IgE, FcRI, and CD32.
Basophil numbers were unaffected by infusions. Eight weeks after
infusions, free IgE levels rose in vivo but did not reach baseline.
Basophil IgE and FcRI rose in parallel with free IgE while CD32
was stable. FcRI densities, measured by acid elution, returned to
80% of baseline, whereas histamine release responses returned to
baseline. Basophils cultured with or without IgE or IgG were analyzed
for expression of IgE, FcRI, and CD32. By 7 days with IgE,
expression of IgE and FcRI rose significantly, whereas cultures
without IgE declined. IgE culture did not effect CD32. IgG culture did
not effect expression of any marker. The present results strongly
suggest that free IgE levels regulate FcRI expression on
basophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI). Binding of multivalent allergens by surface-bound specific
IgE cross-links FcRI and triggers cellular events resulting in the
release of preformed mediators, such as histamine, and newly
synthesized mediators, such as
5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosaraenoic
acid (LTC4) and IL-4 (2). These and other mediators produce
the clinical symptoms of allergic disease and, with repeated allergen
exposure, can promote chronic airway inflammation, the hallmark of
diseases such as allergic rhinitis and asthma. Therefore, the removal
of IgE may offer a specific therapy for control of essentially all
allergic diseases.
Although chronic allergic airway diseases, such as rhinitis and asthma,
have been managed effectively with topical antiinflammatory therapies,
newer treatments directed at IgE have entered human trials. A humanized
monoclonal anti-IgE Ab
(rhumAb-E25)3 has been developed
to complex-free IgE at its receptor binding site, competitively
inhibiting its interaction with FcRI on mast cells and basophils,
thereby preventing sensitization (3). Early clinical trials of
rhumAb-E25 infusions in allergic asthmatics demonstrated suppression of
both early and late responses to inhaled allergen (4, 5). In our hands,
in vivo treatment with rhumAb-E25 led to rapid and dramatic falls in
free serum IgE levels, and of IgE and FcRI receptors on
circulating basophils (6). Reductions in allergen-specific basophil
histamine release were also seen. In more recent work, the presence or
absence of IgE in cultures of peripheral blood basophils determines
whether surface expression of FcRI increases or decreases (7).
The modulation noted in total FcRI surface expression supports
the concept that free serum IgE levels influence the number of FcRI
receptors on the basophil surface. Indeed, a correlation between total
serum IgE levels and the number of high-affinity IgE surface receptors
on peripheral blood basophils has been known for 20 years (8). Thus, if
removal of IgE is related to down-regulation of its receptor on
basophils, conversely, IgE exposure may exert a positive effect on
FcRI receptor expression by circulating basophils. Indeed,
accumulating evidence from cultured mouse mast cells and human
basophils suggests a positive effect of IgE exposure on its own
receptor expression (7, 9, 10).
The aim of the present study was to determine whether
rhumAb-E25-induced reductions in basophil IgE and FcRI
expression, and histamine release responses, were reversible. This was
tested in two ways. First, we examined the kinetics of the reappearance
of free IgE and surface-bound IgE and FcRI on circulating
basophils in individuals completing a total of 46 wk of rhumAb-E25
infusions. Subjects were followed for up to 1 yr by monitoring levels
of free IgE in the serum, as well as basophil surface levels of IgE and
FcRI. At the termination of the follow-up, total IgE receptor
number as well as basophil histamine release to both anti-IgE and
Ag (Dermatophagoides farinae) were examined. Second,
basophils from these subjects obtained within 8 wk of discontinuation
of rhumAb-E25, and therefore expressing markedly reduced levels of
surface IgE and FcRI, were exposed in vitro to IgE in short-term
cultures and examined for effects on basophil IgE and FcRI
surface levels. Using these approaches, we demonstrate a reversal of
rhumAb-E25-induced effects on basophil phenotype both in vitro and in
vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fifteen adult rhinitic subjects with dust mite sensitivity by
skin prick test were enrolled into a phase I safety and tolerance trial
of rhumAb-E25 therapy (Table I
). Nine
subjects also had asthma. Baseline enrollment serum IgE levels
(obtained in 1995) were between 83 and 523 IU/ml (199–1255 ng/ml).
These subjects were randomized to receive two dosing schedules for
rhumAb-E25 based on initial total serum IgE measurements, either 0.015
mg/kg/IU/ml or 0.03 mg/kg/IU/ml, with three initial weekly i.v. loading
infusions followed by maintenance infusions at biweekly intervals (11).
At week 28, subjects were randomized to receive a new maintenance dose
of either 0.005 mg/kg/IU/ml or 0.0015 mg/kg/IU/ml, representing a 3- to
20-fold dose reduction. Patients gave informed written consent for the
rhumAb-E25 study as approved by the Johns Hopkins Bayview Medical
Center Institutional Review Board. Exclusion criteria were as
previously described (6).
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50% of their enrollment level; or 3) the
subjects reached 1 yr post rhumAb-E25 infusions. Buffers
Piperazine-N,N'-bis-2-ethanesulfonic acid (PIPES) obtained from Sigma (St. Louis, MO) was used in a stock buffer composed of 25 mM PIPES containing 110 mM NaCl, 5 mM KCl, and 40 mM NaOH, adjusted to pH 7.3. PAG buffer consisted of PIPES buffer containing 0.003% human serum albumin obtained from Miles Laboratories (Elkhart, IN) and 0.1% glucose; PAGCM buffer consisted of PAG buffer with 1 mM MgCl2 and 1 mM CaCl2; PAG-EDTA buffer consisted of PAG buffer with 1 mM EDTA.
Basophil enrichment
Basophils for culture were enriched from 60 ml of venous blood using a Percoll-based density gradient centrifugation technique as previously described, except that all leukocyte isolations were performed in a sterile manner using 0.45-µm sterile-filtered reagents (12). Basophil purity ranged from 1 to 16% (4 ± 1%, n = 17, mean ± SEM) as enumerated by Alcian blue staining and light microscopic counts (13). Total cell counts and viability (98 ± 0.4%, n = 17, mean ± SEM) were determined by light microscopy and erythrosin B dye exclusion.
Basophils for immediate flow cytometric analysis were isolated from nonsterile 10-ml samples using the same technique (6). For receptor measurements by acid elution and histamine release studies, blood was separated on a single-step Percoll gradient (specific gravity, 1.080), and the interface monolayer was harvested and washed as described (14).
Basophil culture
Enriched basophils were cultured for up to 7 days in Iscove’s modified Dulbecco’s medium from Life Technologies (Gaithersburg, MD) with 2% heat-inactivated FCS, 10 µg/ml gentamicin (Life Technologies) and 10 ng/ml rIL-3 from R&D Systems (Minneapolis, MN) at a final concentration of 1–2 x 106 cells/ml. Cultures were supplemented with or without 50–500 ng/ml myeloma IgE-PS, (15), or 500 ng/ml human IgG (Sigma), containing <0.6 ng/ml IgE as determined by ELISA, Johns Hopkins Dermatology, Allergy, and Clinical Immunology Laboratory. Doses of myeloma IgE used for culture experiments were based on the range of original serum IgE levels of the 11 subjects and preliminary culture studies. Cells were incubated at 37°C in 5% CO2 in 6-well tissue culture plates from Costar (Cambridge, MA) and harvested after 3 or 7 days. After 7 days of culture, viability was 80% ± 4%, n = 17 (mean ± SEM) and total cell recovery was 64% ± 6%, n = 16 (mean ± SEM).
Flow cytometry
Enriched basophil preparations were labeled for direct and
indirect immunofluorescence and flow cytometry in the presence of 4
mg/ml human IgG to minimize nonspecific binding to Fc
R, as
previously described (6). Abs utilized for these studies included an
irrelevant mouse IgG1 control from Coulter (Hialeah, FL);
FITC-conjugated polyclonal goat anti-human IgE and its control
FITC-conjugated normal goat IgG from Kirkegaard and Perry
(Gaithersburg, MD); mouse anti-human FcRI subunit (22E7, IgG1
unaffected by FcRI occupancy, kindly provided by J. Kochan (Roche
Pharmaceuticals, Somerville, NJ) (16) and mouse anti-human FcRII
(CD32) IgG1, mAb IV.3 from Medarex (West Lebanon, NH); or mAB 2E1 from
AMAC (Westbrook, ME). Samples were analyzed on a Coulter
EPICS profile flow cytometer. At least 5000 events per
experimental condition were counted. Data is expressed as net mean
fluorescence intensity (MFI) (actual MFI - MFI of irrelevant IgG
control (4 ± 1, mean ± SEM). Standard microbeads (Sigma)
were run with each group of samples to monitor for day-to-day machine
variability (average bead MFI 49 (47.5, 51.2); 95% confidence
intervals.
Serum-free IgE analysis
Standard total serum IgE assays would capture E25-bound IgE,
therefore, serum-free IgE levels were measured using a solid-phase
ELISA (17) to avoid binding IgE complexed to rhumAb-E25. High-binding
polystyrene plates (Costar) were coated overnight at 2–8°C with 100
ng of human IgE receptor -chain IgG chimera in 100 µl PBS (pH
7.2), washed, and stored frozen. Samples were then analyzed by standard
ELISA methods. After washing, captured IgE was detected with
biotinylated monoclonal anti-human IgE and the color reaction
generated with streptavidin-ß-galactosidase and
4-methylumbelliferyl-ß-D-galactoside. The detection limit
of the assay was 0.8 ng/ml with an upper limit of 226 ng/ml. For
samples containing <40 ng/ml of total E25, dilutions of serum samples
were possible for free IgE measurements. A second assay for a higher
range of free IgE detection was the same, except plates were coated in
carbonate buffer (pH 9.6) and processed with a streptavidin-horseradish
peroxidase and
o-phenylenediamine/H2O2 (other assay
differences, e.g., diluent, not discussed). The higher range assay had
a range of 24–396 ng/ml. The high-range and low-range assay differ
systematically in quantitation, with the high-range assay giving higher
free IgE estimates. Exploratory analysis showed that the percent
difference in quantitation was approximately constant over the assay
range, ±3.6% higher than the low-range assay (estimate ± SE).
To compare high- and low-range assay results in this study, the
high-range assay results were multiplied by an adjustment factor of
1/1.600 = 0.625.
Measurements of open and occupied FcRI on basophils
To determine both the total and unoccupied receptor densities, isolated mononuclear cells were first sensitized with benzylpenicilloyl (BPO)-specific IgE (5 µg/ml) for 20 min at 37°C in RPMI 1640 medium (Life Technologies) containing 1 mM EDTA and 10 µg/ml heparin (6). Previous studies demonstrated that sensitization with this concentration of BPO-specific IgE for this length of time effectively saturates unoccupied receptors and, therefore, allowed measurement of total receptors (by measuring the total IgE eluted from saturated cells) as well as the measurement of unoccupied receptor density (by measuring the total BPO-specific IgE eluted from saturated cells; see below) (18). After washing once, the cells were layered over 1 ml of EDTA-chelated FCS (5 mM EDTA in heat-inactivated FCS) and centrifuged to separate the cells from their diluted sensitization buffer. The cells were further washed twice in PAG, then resuspended in PAG buffer and incubated for 60 min at 37°C. After centrifugation, the pellets were resuspended in 1 ml of saline. After a final centrifugation, duplicate samples were removed for cell counts. Supernatants from sensitized cells are also analyzed to ensure a lack of IgE carry-over from the sensitization step.
The pellets were resuspended in ice-cold acetate buffer (pH 3.7) and incubated in an ice bath for 10 min. After a brief centrifugation (15,000 x g), the supernatant was removed and neutralized with 1 N NaOH. The eluted IgE was measured in either a total IgE RIST (total receptor measurement) or BPO-RAST (unoccupied receptors) (17). Cell counts (Alcian blue-positive cells) obtained before elution allowed a calculation of the receptor density to be made (the amount of IgE measured by RIST or RAST divided by cell count with the result expressed as IgE molecules per basophil).
Histamine measurements
The histamine content of whole blood leukocytes was analyzed by automated fluorometric analysis of perchloric acid lysates of 100-µl aliquots of whole blood as performed previously (6, 19).
Aliquots of mononuclear cells destined for receptor analysis were also analyzed for maximal histamine release by examination of dose response curves for polyclonal goat anti-human-IgE Ab (0.01–10 µg/ml). In addition, two doses of D. farinae (10 and 0.5 PNU/ml) from Miles (Spokane, WA), previously shown to be optimal and suboptimal for release, were included for Ag-specific basophil histamine release (6). Briefly, cells were suspended in PAGCM and challenged with the stimulus for 45 min at 37°C at a final volume of 1 ml. All reactions were performed in duplicate and stopped by centrifugation, and the supernatant was removed for histamine analysis.
Statistical methods
All values are mean ± SD, unless otherwise noted. A nonparametric Wilcoxon signed rank statistic was used to determine differences in the data sets. Correlations were calculated using simple regression analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 11 of the original 15 subjects participated in the post
rhumAb-E25 portion of the study. Of these, four subjects were
terminated from the follow-up study by meeting the first criteria, four
others the second criteria, and three subjects completed the entire
1-yr follow-up period (see Materials and Methods). During
the follow-up period after the final infusion with rhumAb-E25, subjects
slowly regained levels of detectable serum-free IgE. Shown in Fig. 1 is the rise of free serum IgE in the 11
subjects who completed the follow-up period. Mean free IgE increased
from 20 ± 3 ng/ml 1 h after last rhumAb-E25 infusion (time
0) to 94 ± 17 ng/ml at 8 wk post rhumAb-E25, reaching 16% of the
pre-rhumAb-E25 infusion mean of 583 ± 106 ng/ml. At the
termination from the follow-up portion of the study, serum-free IgE
reached different levels depending on the reason for termination (Table II). Subjects who received the highest
dosing regimen of rhumAb-E25 (0.03 mg/kg/IU/ml the first 28 wk and
subsequent 6-fold dose reduction for the remaining 18 wk) had the
longest follow-up in comparison to other dosing regimens (343 ±
13 days, n = 4 (subjects 5, 7, 9, 11; Table II) vs
218 ± 32 days, n = 7).
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RI, and FcRII
expression
As shown in Table III, the relative
percentage of basophils in mononuclear cell preparations as measured by
Alcian blue staining during and after the rhumAb-E25 infusion period
remained stable. Likewise, the histamine content of whole blood lysates
remained stable during and after rhumAb-E25 infusions. Thus, rhumAb-E25
infusions did not alter the percentage or the average histamine content
of circulating basophils.
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RI on
basophils also increased (Fig. 2).
Basophils obtained within 4 wk of the last rhumAb-E25 infusion
demonstrated either low (n = 9) or undetectable
(n = 2) surface labeling for IgE (Fig. 2A).
The limit of detection is 
8,000–10,000 surface IgE molecules (20).
During the observation period in this study, IgE surface intensity
returned to baseline levels in only a single subject (subject 3, 24 wk
post rhumAb-E25 after a 50% return of free IgE). Median MFI of
surface IgE staining rose significantly by 8 wk after the last
rhumAb-E25 infusion (p < 0.01,
n = 11, 2.7-fold increase), and continued to rise, so
that by the time of termination, a median 4.4-fold increase had
occurred (p < 0.01, n = 11).
These termination values represent 47% of the enrollment,
pre-rhumAb-E25 infusion values while free serum IgE levels had reached
an average of 62% (n = 8, three subjects
indeterminable) of the enrollment values (6).
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RI expression was also noted (Fig. 2
B) with a median rise in MFI from 60 at day 0 to 118 by day
56 (p < 0.01, n = 11,
2-fold increase). By the end of the study, median expression reached
266 MFI (p = 0.001, n = 11,
4.4-fold increase). Unfortunately, enrollment levels of FcRI were
not analyzed (6) so no comparison is available. In contrast, expression
of another surface Ig receptor, FcRII (CD32), was not significantly
altered during the same period (data not shown).
In support of the hypothesis that free serum IgE levels influence
basophil FcRI expression, significant correlations were seen
between free serum IgE levels and basophil surface phenotype at three
time points during the follow-up period (Fig. 3). Receptor occupancy appeared to be
fairly consistent at these time points given that the intensity of
surface IgE was correlated to the intensity of IgE receptor
(r2 = 0.49, p = .0001).
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Shown in Fig. 4 are the results of
additional basophil receptor numbers as measured by the lactic-acid
elution method. Fig 4A shows mean pretreatment levels of
total basophil receptors to be 
230,000 per basophil. After 12 wk of
rhumAb-E25 infusion, mean receptor levels were dramatically reduced to
<10,000 receptors per basophil, as previously described (6). By the
time of discontinuation of rhumAb-E25 (i.e., 18 wk after institution of
rhumAb-E25 dosage reductions; see Materials and Methods),
receptors had risen to nearly 35,000 per basophil. At the termination
of the follow-up study (see Table II), the receptor numbers had almost
returned to pretreatment levels (214,000 per basophil).
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, B and
C. The mean basophil histamine release responses to an
optimal dose of D. farinae (10 PNU/ml) were reduced by
90% after 12 wk of rhumAb-E25 treatment, whereas
anti-IgE-mediated release was reduced to a lesser extent (40%),
as previously reported (6). However, by the final rhumAb-E25 infusion
(46 wk), the 3-fold rise in receptor number (Fig. 4A) and
rising free IgE levels (Fig. 1) were associated with an almost complete
return of D. farinae-specific histamine release and a
complete return of anti-IgE-induced histamine release. Effect of culture with IgE or IgG on basophils from rhumAb-E25-treated subjects
Studies of human basophils have demonstrated that IgE induces
up-regulation of FcRI (7). Given the markedly reduced IgE surface
expression present on basophils at the completion of rhumAb-E25
infusions, we took advantage of this unique opportunity to see whether
basophils from these subjects would display similar regulation of
FcRI by IgE. Basophils obtained within 8 wk of discontinuation of
rhumAb-E25 were cultured with IgE (or IgG, as a control) and effects on
basophil surface expression of IgE, FcRI, and FcRII (CD32, as
a control) were examined. No significant enhancement of surface IgE and
FcRI expression was detectable at 3 days of culture with myeloma
IgE (data not shown); however, 7 days of culture with myeloma IgE
resulted in significant 1.6- to 2-fold enhancement of basophil
FcRI and surface IgE staining, respectively (Fig. 5). The enhancement in both surface IgE
and FcRI expression was dose-dependent in that parallel cultures
using 500 ng/ml demonstrated greater enhancement than 50 ng/ml IgE
cultures (n = 5–6; data not shown). Culture in medium
alone led to a slight but significant decline in surface IgE, whereas
FcRI did not change. Basophils cultured in the presence of 500
ng/ml IgG resembled basophils cultured with medium alone in that no
significant increases in expression of IgE or FcRI were observed.
As a control, expression of another Ig receptor on basophils FcRII
(CD32) was also assessed and did not change significantly under either
of the Ig culture conditions (data not shown). Cell recovery and
viability did not differ among each of the culture conditions.
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B) was related to starting
FcRI expression (Fig. 6). Basophils
cultured within 2 wk of rhumAb-E25 completion, and hence lower surface
expression of FcRI, showed larger increases than later cultures,
as previously demonstrated (7). Increases ranged from 15- to 0.7-fold
for starting FcRI densities ranging from 15 to 250 MFI. Basophils
cultured within 2 wk of completion of rhumAb-E25 rose from a median of
50 on day 0 to a median of 122 on day 7 (n = 9,
p = .004, 2.4-fold increase). A single culture using
basophils from a patient in the midst of rhumAb-E25 infusions (week 36
of 46) yielded a 3.8-fold rise over the 7 day culture period (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI. The relative
strength of the correlation (r2 > 0.5) supports the
existence of a relationship between serum IgE levels and basophil
phenotype for IgE and FcRI density. A separate measurement of
total basophil receptor number (acid elution) also demonstrated a
return in basophil FcRI receptor levels to nearly pretreatment
levels by the termination endpoints from the follow-up study. Basophil
surface enhancement of IgE and FcRI after rhumAb-E25 occurred
without effects on numbers of isolated basophils from the circulation.
Furthermore, whole blood histamine content, another marker of blood
basophil presence, also remained stable during and after rhumAb-E25
infusions. The effect on basophil surface phenotype was selective,
because levels of an additional basophil Ig receptor (FcRII, CD32)
remained unaltered during the same period. Thus, discontinuation of
rhumAb-E25 therapy led to a reversal of its effects on both free serum
IgE levels and basophil surface expression of IgE and FcRI.
Unlike the situation after 3 mo of rhumAb-E25 treatment when
Ag-specific histamine release was reduced by 90% (6), by the time
of final rhumAb-E25 infusion, Ag-specific release was nearly restored
despite only a modest rise in receptor number. Thus, functional effects
of receptor down-regulation by rhumAb-E25 were restored at receptor
levels well below pretreatment levels. This restoration can be
interpreted as a consequence of both receptor repopulation as well as
rising free serum IgE levels seen in subjects after conclusion of
rhu-MAb-E25. Given that the threshold number of IgE-cross-links by Ag
needed to cause maximal basophil histamine release has been reported to
be several thousand, this setpoint appears to have been partially
reestablished as a result of the rhumAb-E25 dose reduction protocol.
Furthermore, Ag-induced histamine responses were fully restored within
weeks of rhumAb-E25 discontinuation. The clinical implications of this
phenomena of restored basophil histamine release in terms of therapy
with rhumAb-E25 remains to be established.
In vitro experiments with basophils isolated from former rhumAb-E25
recipients and exposed in culture to myeloma IgE also showed
enhancement of surface expression of IgE and FcRI receptors. The
low level of FcRI and surface IgE expression induced in vivo on
basophils from rhumAb-E25-treated subjects was nearly doubled by
short-term in vitro culture with myeloma IgE. Parallel cultures with
equal amounts of IgG or medium alone failed to show enhancement,
supporting the specificity of IgE in altering basophil surface
expression. The increase in the density of cell surface FcRI in
vitro was more rapid than observed in vivo, suggesting that 1) there is
no alteration in the ability of the basophils to up-regulate FcRI
since these results are similar to those found with rhumAb-E25-naive
donors in in vitro studies, and 2) that the slower rise in vivo
probably results from the slower rise in free IgE levels. In vitro
cultures also showed dose dependence on IgE for high-affinity receptor
enhancement. As before, starting levels of receptor expression appear
to predict the degree of receptor enhancement. The lower the initial
starting level of FcRI, the greater the resulting enhancement
seen in basophil cultures as shown in basophils cultured from
rhumAb-E25-naive donors (6), where the fold increase of FcRI
expression after 1 wk of culture with myeloma IgE (2.5-fold) was
similar to that observed in Fig. 5 in our basophil cultures from E25
recipients. Thus, free IgE levels appear to regulate the levels of
FcRI and IgE expression on cultured human blood basophils
extending the activity of free IgE to both in vitro and in vivo
conditions.
Evidence for receptor regulation by free IgE levels is not limited to
human basophil studies. Work in other cultured basophil systems,
including basophil cell lines (21), or mouse bone marrow basophil
cultures (21), in vitro and in vivo mast cell studies in the mouse (9),
and cultured human fetal liver mast cells (22), all have shown FcRI
regulation by ambient IgE levels. However, this is the first report
that human peripheral blood basophils demonstrate up-regulation of the
high-affinity receptor in association with rising serum levels of free
IgE in vivo.
The mechanism for this IgE-dependent effect, whether direct or
indirect, remains unclear. Experiments using protein synthesis
inhibitors in murine mast cells (9) suggested that up-regulation of the
high-affinity receptor could not be completely explained by preformed
receptors that are stabilized under the influence of free IgE, as was
suggested by studies using rat basophilic leukemia cells (23).
Furthermore, the kinetics of receptor loss seem to follow a time course
measured in days, despite a rapid fall in free IgE levels that occurred
over hours, such as with the first infusions of rhumAb-E25 Ab.
Conversely, in vitro restoration of the basophil surface receptors
required days to repopulate both IgE and FcRI, without detectable
enhancement present at 3 days (as measured by the limits of flow
cytometer).
A similar scenario could be invoked for the in vivo results after decay of excess rhumAb-E25; however, the in vivo enhancement was complicated by levels of free IgE that were the net effect of ongoing IgE synthesis in the face of declining rhumAb-E25 levels. Unlike murine systems demonstrating a suppression of IgE synthesis by treatment with anti-IgE mAbs (24), the in vivo experience with rhumAb-E25 did not appear to suppress on-going IgE synthesis. Whether the subjects free IgE level will return to baseline levels is not yet established.
The in vitro up-regulation of receptor occurred in the presence of
rIL-3, which was necessary to maintain basophil viability for 1 wk.
IL-3 is known to prime basophils for inflammatory activities (25), as
well as support bone marrow precursor development toward a basophilic
lineage (26). However, in the presence of IL-3 alone, we failed to
detect basophil FcRI receptor enhancement and, in fact, observed
a slight but consistent loss of receptor. Furthermore, the specificity
for IgE to cause receptor enhancement in cultures suggested that
binding of IgE to the basophil is needed to achieve receptor induction.
Recent in vitro studies by MacGlashan (6) show that a mixture of an
excess of an anti-IgE mAb with IgE in basophil cultures prevented
basophil receptor up-regulation, supporting a need for IgE binding to
FcRI for enhancement. While others have shown a role for IL-4 in
further enhancing induction of FcRI (22, 27, 28), this was not
examined in our studies.
Given the mixed leukocyte composition of our culture preparations, we
needed to exclude expression and measurement of high-affinity receptor
on contaminating cells. Other circulating leukocytes such as monocytes
may bear high-affinity IgE receptors in certain atopic conditions (29, 30). The use of gating by cellular scatter characteristics allowed
exclusion of most monocytes from FcRI analysis by flow cytometry.
Furthermore, preliminary experiments designed to preferentially detect
monocyte FcRI expression enhancement under identical culture
conditions have failed to demonstrate such an IgE-dependent effect.
Previous reports of monocyte FcRI expression (29, 30) have been
generally limited to individuals with atopic dermatitis and at
considerably lower levels of intensity (2 logs) than basophil as
measured by flow cytometry.
Infusions of an IgE-binding mAb in human subjects have reduced basophil
FcRI expression and histamine release responses to Ag, both of
which are reversed after discontinuing infusions. Restoration of
Ag-triggered histamine release responses occurred more rapidly than
receptor number and implies a lower threshold for basophil activation
through Ag interaction with specific IgE bound to its receptor.
Furthermore, basophil FcRI receptor enhancement occurred under
the influence of IgE, both in vivo and in vitro, and suggests a
regulation by free IgE of basophil receptor expression. Further studies
to examine the exact mechanism of IgE-dependent regulation of the
high-affinity receptor on human basophils are needed.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce S. Bochner, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address:
3 Abbreviations used in this paper: rhumAb-E25, a humanized monoclonal anti-IgE Ab; MFI, mean fluorescence intensity; BPO, benzylpenicilloyl.
Received for publication October 9, 1998. Accepted for publication February 12, 1999.
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  C. B. Mathias, E.-J. Freyschmidt, B. Caplan, T. Jones, D. Poddighe, W. Xing, K. L. Harrison, M. F. Gurish, and H. C. Oettgen IgE Influences the Number and Function of Mature Mast Cells, but Not Progenitor Recruitment in Allergic Pulmonary Inflammation J. Immunol., February 15, 2009; 182(4): 2416 - 2424. [Abstract] [Full Text] [PDF]
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 G. Pelaia, T. Renda, P. Romeo, M. T. Busceti, and R. Maselli Review: Omalizumab in the treatment of severe asthma: efficacy and current problems Therapeutic Advances in Respiratory Disease, December 1, 2008; 2(6): 409 - 421. [Abstract] [PDF]
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 J. M. Langdon, J. T. Schroeder, B. M. Vonakis, A. P. Bieneman, K. Chichester, and S. M. MacDonald Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models J. Leukoc. Biol., October 1, 2008; 84(4): 1151 - 1158. [Abstract] [Full Text] [PDF]
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 B. M. Vonakis, D. W. MacGlashan Jr, N. Vilarino, J. M. Langdon, R. S. Scott, and S. M. MacDonald Distinct characteristics of signal transduction events by histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced priming and activation of human basophils Blood, February 15, 2008; 111(4): 1789 - 1796. [Abstract] [Full Text] [PDF]
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 B. M. Jensen, M. A. Beaven, S. Iwaki, D. D. Metcalfe, and A. M. Gilfillan Concurrent Inhibition of Kit- and Fc{epsilon}RI-Mediated Signaling: Coordinated Suppression of Mast Cell Activation J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 128 - 138. [Abstract] [Full Text] [PDF]
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D. W. MacGlashan Jr. Endocytosis, recycling, and degradation of unoccupied Fc{epsilon}RI in human basophils J. Leukoc. Biol., October 1, 2007; 82(4): 1003 - 1010. [Abstract] [Full Text] [PDF]
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 E. Hamelmann The rationale for treating allergic asthma with anti-IgE Eur. Respir. Rev., September 1, 2007; 16(104): 61 - 66. [Abstract] [Full Text] [PDF]
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G. Gomez, S. Jogie-Brahim, M. Shima, and L. B. Schwartz Omalizumab Reverses the Phenotypic and Functional Effects of IgE-Enhanced Fc{epsilon}RI on Human Skin Mast Cells J. Immunol., July 15, 2007; 179(2): 1353 - 1361. [Abstract] [Full Text] [PDF]
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 R. C. Strunk and G. R. Bloomberg Omalizumab for asthma. N. Engl. J. Med., June 22, 2006; 354(25): 2689 - 2695. [Full Text] [PDF]
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T. Kubota, K. Mukai, Y. Minegishi, and H. Karasuyama Different Stabilities of the Structurally Related Receptors for IgE and IgG on the Cell Surface Are Determined by Length of the Stalk Region in Their {alpha}-Chains. J. Immunol., June 1, 2006; 176(11): 7008 - 7014. [Abstract] [Full Text] [PDF]
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 G. Hanf, O. Noga, A. O'Connor, and G. Kunkel Omalizumab inhibits allergen challenge-induced nasal response Eur. Respir. J., March 1, 2004; 23(3): 414 - 419. [Abstract] [Full Text] [PDF]
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S. Kubo, T. Nakayama, K. Matsuoka, H. Yonekawa, and H. Karasuyama Long Term Maintenance of IgE-Mediated Memory in Mast Cells in the Absence of Detectable Serum IgE J. Immunol., January 15, 2003; 170(2): 775 - 780. [Abstract] [Full Text] [PDF]
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 M. P. HORN, J. M. PACHLOPNIK, M. VOGEL, M. DAHINDEN, F. WURM, B. M. STADLER, and S. M. MIESCHER Conditional autoimmunity mediated by human natural anti-Fc{varepsilon}RI{alpha} autoantibodies? FASEB J, October 1, 2001; 15(12): 2268 - 2274. [Abstract] [Full Text] [PDF]
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S. Kubo, K. Matsuoka, C. Taya, F. Kitamura, T. Takai, H. Yonekawa, and H. Karasuyama Drastic Up-Regulation of Fc{epsilon}RI on Mast Cells Is Induced by IgE Binding Through Stabilization and Accumulation of Fc{epsilon}RI on the Cell Surface J. Immunol., September 15, 2001; 167(6): 3427 - 3434. [Abstract] [Full Text] [PDF]
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T. A. Borkowski, M.-H. Jouvin, S.-Y. Lin, and J.-P. Kinet Minimal Requirements for IgE-Mediated Regulation of Surface Fc{epsilon}RI J. Immunol., August 1, 2001; 167(3): 1290 - 1296. [Abstract] [Full Text] [PDF]
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 M. Yamaguchi, K. Hirai, A. Komiya, M. Miyamasu, Y. Furumoto, R. Teshima, K. Ohta, Y. Morita, S. J. Galli, C. Ra, et al. Regulation of mouse mast cell surface Fc{{varepsilon}}RI expression by dexamethasone Int. Immunol., July 1, 2001; 13(7): 843 - 851. [Abstract] [Full Text] [PDF]
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N. Kambe, M. Kambe, J. P. Kochan, and L. B. Schwartz Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes Blood, April 1, 2001; 97(7): 2045 - 2052. [Abstract] [Full Text] [PDF]
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B. Albrecht, M. Woisetschlager, and M. W. Robertson Export of the High Affinity IgE Receptor From the Endoplasmic Reticulum Depends on a Glycosylation-Mediated Quality Control Mechanism J. Immunol., November 15, 2000; 165(10): 5686 - 5694. [Abstract] [Full Text] [PDF]
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C. L. Kepley, L. Youssef, R. P. Andrews, B. S. Wilson, and J. M. Oliver Multiple Defects in Fc{epsilon}RI Signaling in Syk-Deficient Nonreleaser Basophils and IL-3-Induced Recovery of Syk Expression and Secretion J. Immunol., November 15, 2000; 165(10): 5913 - 5920. [Abstract] [Full Text] [PDF]
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P. J. Barnes Anti-IgE Antibody Therapy for Asthma N. Engl. J. Med., December 23, 1999; 341(26): 2006 - 2008. [Full Text]
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