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RI
by Human Eosinophils1
Department of Medicine, Division of Clinical Immunology, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R have been detected on human eosinophils,
levels varied from moderate to extremely low or undetectable depending
on the donor and methods used. We have attempted to resolve the
conflicting data by measuring levels of IgE, FcRI, and FcRII in
or on human eosinophils from a variety of donors (n
= 26) and late-phase bronchoalveolar lavage fluids
(n = 5). Our results demonstrated little or no cell
surface IgE or IgE receptors as analyzed by immunofluorescence and flow
cytometry. Culture of eosinophils for up to 11 days in the presence or
absence of IgE and/or IL-4 (conditions that enhance FcR on other
cells) failed to induce any detectable surface FcR. However,
immunoprecipitation and Western blot analysis of eosinophil lysates
using mAb specific for FcRI
showed a distinct band of
approximately 50 kDa, similar to that found in basophils. Western
blotting also showed the presence of FcR 
-chain, but no FcRIß.
Surface biotinylation followed by immunoprecipitation again failed to
detect surface FcRI, although surface FcR was easily detected.
Since we were able to detect intracellular FcRI, we examined its
release from eosinophils. Immunoprecipitation and Western blotting
demonstrated the release of FcRI into the supernatant of cultured
eosinophils, peaking at approximately 48 h. We conclude that
eosinophils possess a sizable intracellular pool of FcRI that is
available for release, with undetectable surface levels in a variety of
subjects, including those with eosinophilia and elevated serum IgE. The
biological relevance of this soluble form of FcRI remains to be
determined. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Helminthic infections and allergic diseases share not only peripheral blood and tissue eosinophilia, but also high levels of circulating IgE. Although the pathogenic role of IgE in immediate hypersensitivity reactions is well characterized, the significance of the IgE response to parasitic infections is poorly understood. Recent studies document significant correlations between high levels of specific IgE against the parasite and a lower rate of reinfection (6, 7, 8), suggesting a protective role for IgE against parasitic infections. The coexistence of high levels of IgE and eosinophilia in both allergic and parasitic diseases suggests that IgE may directly interact with and stimulate eosinophils.
The high affinity IgE receptor (FcRI) is a membrane glycoprotein
composed of an subunit, a ß subunit, and dimeric subunits
(9). The -chain contains the extracellular IgE-binding domain (10),
while the ß- and -chains are felt to serve as intracellular signal
transducers (11, 12). In human cells, expression of FcRI requires
the presence of the - and -chains, but not the ß-chain (13).
FcRI is primarily expressed by mast cells and basophils (14). Recent
studies demonstrate, however, that FcRI may also be expressed by
epidermal Langerhans cells (15) and monocytes from some donors (16).
Although cell surface expression of FcRI has also been described on
eosinophils from patients with hypereosinophilic syndromes (17),
conflicting results have been obtained depending on the donor as well
as the methods used. For example, while immunohistochemistry or
immunocytochemistry methods often find high levels of the -chain
(18), cell surface levels of FcRI, as analyzed by immunofluorescence
and flow cytometry, were extremely low or undetectable (16). In
addition, a potential role on eosinophils for other IgE-binding
structures, such as the low affinity IgE receptor (CD23, FcRII) and
Mac-2, has been suggested (19). To enhance our understanding of
eosinophil biology and its relationship to IgE in the pathogenesis of
inflammatory diseases, we have attempted to resolve the existing
conflicting data.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For most experiments, eosinophils were purified, as previously
described, using Percoll density centrifugation (specific gravity
= 1.090), followed by negative selection using anti-CD16 Abs to
remove neutrophils from peripheral blood of a variety of donors,
including subjects with mild allergic rhinitis and/or asthma (donors
3–11, 12, 14, 17–21), as well as those with diverse hypereosinophilic
conditions (Table I
) (20, 21).
Eosinophils were also isolated and enriched from bronchoalveolar lavage
(BAL)3 fluid collected 20 h
after segmental allergen challenge of allergic asthmatics (donors 10,
12–15) using a similar protocol (21, 22). Eosinophil purity in both
types of preparations was always >98%, with neutrophils being the
only contaminating cells. For some experiments, eosinophils were
identified and examined in anticoagulated blood of normals and other
subjects without enrichment using dual color immunofluorescent methods
(Refs. 23 and 24, and see below).
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Peripheral blood basophils were isolated by elutriation and Percoll density gradients of leukopheresis packs to >60% purity (25).
Cell culture
Eosinophils and the Jurkat T cell lymphoma cell line (American
Type Culture Collection, Manassas, VA) were maintained at 37°C in
RPMI 1640 media supplemented with 5% FBS and 1%
penicillin/streptomycin (Life Technologies, Gaithersburg, MD). Jurkat
cells were passaged every 3 to 4 days. All eosinophil cultures
contained 10 ng/ml IL-5 (R&D Systems, Minneapolis, MN) to maintain
viability, which was always >75%, as assessed by dye exclusion under
light microscopy using Erythrocin B (26). Some eosinophil cultures were
supplemented with either 1 µg/ml IgE (PS myeloma, a gift of Dr. T.
Ishizaka, Johns Hopkins University, Baltimore, MD), 10 ng/ml IL-4
(Genzyme, Cambridge, MA), or a combination of both for up to 11
days; these conditions have previously been shown to facilitate
increases in FcRI protein on human basophils (27) and mouse mast
cells (28).
Flow cytometry
Two different methods were used to examine expression of FcR
or cell surface IgE by direct or indirect immunofluorescence and flow
cytometry. Briefly, in the first protocol (29), cells were incubated
for 30 min at 4°C in PBS containing 0.1% BSA and 4 mg/ml human IgG
with saturating concentrations of receptor-specific Ab or an equivalent
concentration of an irrelevant isotype-matched control Ab. For this
study, we used a FITC-conjugated polyclonal goat anti-human IgE and
its FITC-conjugated polyclonal normal goat IgG control (Kirkegaard &
Perry, Gaithersburg, MD) or the following IgG1 mouse anti-human
mAbs: FcRI -chain (22E7 and 15A5, kindly provided by Dr. J.
Kochan, Hoffman-La Roche, Nutley, NJ), FcRII (CD23) (9P.25;
Immunotech, Westbrook, ME), CD44 (J173; Immunotech), CD69 (TP1/55.3.1;
Biosource, Camarillo, CA), and an IgG1 isotype control mAb from Coulter
(Hialeah, FL). Cells were washed and then incubated with 1/100
dilutions of R-phycoerythrin-conjugated F(ab')2 goat
anti-mouse IgG Ab (Biosource) for 30 min at 4°C in the dark,
washed, and fixed in 1% paraformaldehyde in PBS.
In the second protocol, eosinophils from donors 1, 2, 12–16, and
22–26 (Table I
) were distinguished from neutrophils in whole blood
samples in which eosinophil purity was much lower (23, 24). In brief,
hypotonic lysis was first performed, then cells were labeled with FITC
anti-CD9 mAb (Research Diagnostics, Flanders, NJ; which recognizes
eosinophils, but not neutrophils (30)) in the presence of excess normal
mouse IgG after labeling with the unconjugated mAb and PE
anti-mouse IgG. Cells were then washed and fixed in 1%
paraformaldehyde in PBS. Light scatter analysis was then used to
isolate granulocytes, and window gating was performed to identify the
CD9+ population. For both protocols, at least 1000 cells
were analyzed with a Coulter EPICS Profile II flow cytometer.
Immunoprecipitation and Western blot analysis
Cells were lysed for 20 min on ice with Triton lysis buffer (20
mM Tris (pH 7.4), 100 mM NaCl, 10 mM
Na4P2O7, 2 mM EDTA, 50 mM NaF; 1%
Triton X-100; 200 mM PMSF; 1 mM NaVO4; 1 mM each leupeptin,
aprotinin, and pepstatin A (Sigma, St. Louis, MO)). Insoluble cell
debris was removed by centrifugation (15,000 x g, 5
min, 4°C). For Western blot analysis of whole cell extracts, lysates
from 2 x 106 cells were boiled for 5 min in SDS
sample buffer (2% SDS, 50 mM Tris, pH 6.8, 100 mM DTT, 0.1%
bromophenol blue, 10% glycerol). For immunoprecipitation experiments,
whole cell extracts from 5 x 106 cells were incubated
for 2 h at 4°C with mAbs to the FcRI -chain (22E7 or 15A5)
bound to protein G-Sepharose, or with a FcR-specific polyclonal
rabbit antiserum (934, generously provided by Dr. J.-P. Kinet, Harvard
University, Cambridge, MA) bound to protein A-Sepharose beads
(Pharmacia Biotech, Uppsala, Sweden). The beads were washed four to
five times in lysis buffer, 20 µl sample buffer was added, and the
samples were boiled for 5 min. Lysates or immunoprecipitates were
separated by SDS-PAGE electrophoresis (31), and proteins were
transferred to polyvinylidene difluoride membrane (Bio-Rad, Hercules,
CA). Membranes were blocked in PBS containing 4% BSA overnight at
4°C. The membranes were immunoblotted with Ab 22E7 (FcRI), 15A5
(FcRI), 976 (FcRIß-specific polyclonal rabbit antiserum,
generously provided by Dr. J.-P. Kinet, Harvard University), or 934
(FcR). Membranes were then washed three times for 10 min in
TBS-Tween (0.2%) before incubating with an appropriate HRP-conjugated
secondary Ab for 45 min. Membranes were subjected to three additional
washes before proteins were visualized by enhanced chemoluminescence
(Amersham, Burlington Heights, IL).
Surface biotinylation and immunoprecipitation
After washing away Tris-related amines with PBS, intact cells were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin, according to the manufacturer’s instructions (Pierce, Rockford, IL). The reaction was stopped with 1 M NH4Cl, and cells were washed in TBS and lysed, as described above. Proteins were immunoprecipitated and separated, as described above. Biotinylated proteins were visualized using HRP-conjugated streptavidin, followed by enhanced chemoluminescence. The membrane was then stripped with 7 M guanidine-HCl for 20 min at room temperature and reprobed by Western blot, as described above.
Analysis of supernatants
Eosinophils were cultured with IL-5 (10 ng/ml) for up to 8 days.
At various time points, supernatant aliquots were collected and
centrifuged to remove any intact cells present. Sample buffer was
added, and the sample was boiled for 5 min. Western blotting was
performed as described above. For immunoprecipitation experiments,
3 x 106 eosinophils were lysed and FcRI
immunoprecipitated immediately after purification. A separate aliquot
of 3 x 106 eosinophils from the same donors was
placed in culture with IL-5 (10 ng/ml) for 24 h. At that time,
cells were separated from the supernatant by centrifugation. Both
samples were then boiled for 5 min with sample buffer.
Immunoprecipitation and Western blotting were conducted as described
above.
Statistical analysis
All data are expressed as mean ± SEM. Differences between groups were assessed by ANOVA. Statistical significance was reached at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R on purified eosinophils and
eosinophils labeled in whole blood
Expression of FcR on the surface of peripheral blood or
late-phase BAL eosinophils was analyzed by indirect immunofluorescence
and flow cytometry. There was no significant cell surface expression of
FcRI on eosinophils from normal donors or subjects with any of
the eosinophilic conditions examined, as evidenced by staining with the
mAb 22E7 (Fig. 1), which recognizes
FcRI regardless of its state of occupancy by IgE. The 22E7 mAb has
been shown to stain human basophils from allergic donors, in which
values of 10–100-fold greater than IgG control are typically observed
(32). In all but 1 of the 31 eosinophil preparations tested, mean
fluorescence intensity (MFI) was <1.5-fold that seen with the
irrelevant IgG control mAb. A complete lack of detectable staining was
obtained with all purified eosinophil preparations using a
FITC-conjugated polyclonal anti-IgE antiserum and a CD23 (FcRII)
mAb (data not shown). Previous studies using impure basophils (<5%)
indicated that cell surface IgE densities needed to be >7000 to be
detected by flow cytometry (29). More recently, we reevaluated this
sensitivity of the instrument when using purified human basophils or
RBL cells, and found that densities of 1000–2000 cell surface IgE or
FcRI molecules could be discriminated from control antibody
cytometric distributions ((33) and unpublished observations).
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R
Eosinophils were cultured with IL-5 (10 ng/ml) in the presence or
absence of IgE (1 µg/ml), IL-4 (10 ng/ml), or a combination of both,
for up to 11 days, conditions that have previously been shown to lead
to an increase in cell surface expression of FcRI on basophils and
mast cells and an up-regulation of FcRI -chain mRNA levels in
human eosinophils (27, 28, 34, 35). Surface expression of FcR and
IgE was analyzed before culture and at 6 and 11 days of culture, with
mAb 22E7 to the FcRI -chain, the FITC-conjugated polyclonal
anti-IgE antiserum, and a mAb to CD23 (FcRII). No significant
increase in the levels of expression of any of the FcR markers was
seen for the different donors at any of the time points studied (Fig. 2 and data not shown). We also performed
immunofluorescence and flow cytometry at earlier time points, and again
were unable to observe FcRI protein, as detected by mAb 22E7, on
the surface of the eosinophils at 24 or 48 h of culture (1.1
± 0.1-fold and 0.8 ± 0.1-fold MFI (n = 3),
respectively). However, other surface markers, such as CD44 and CD69
(24), were highly expressed and appropriately up-regulated on the
surface of the eosinophil (data not shown).
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RI -chain protein in eosinophil lysates
Although we did not detect significant cell surface expression of
FcRI, previous reports indicate that eosinophils contain FcRI
-chain protein (as determined immunohistochemically) and
mRNA-encoding FcRI -chain (17, 18, 34, 36, 37, 38, 39). -chain protein
was immunoprecipitated from purified blood and BAL eosinophil lysates
with mAb 22E7; an irrelevant IgG control mAb was also used
(n = 5). A broad prominent band at approximately 50 kDa
(the known molecular mass of FcRI -chain) was easily detectable
in eosinophil lysates from a variety of donors (Fig. 3A, lane 1). In two
of five experiments (including the one displayed in Fig. 3A,
lane 1), a second fainter band of slightly higher relative
m.w. was also detected. FcRI -chain was not immunoprecipitated
with the control mAb and was also not detected in Jurkat T cell lysates
(Fig. 3A). Furthermore, identical results were obtained when
using a different immunoprecipitating mAb (15A5) reactive against
another epitope of the -chain (data not shown). In matching
experiments, basophils expressed a protein of similar, although
slightly lower, molecular mass (Fig. 3B), subtle differences
likely due to differences in glycosylation. Thus, although FcRI
-chain was not detected by flow cytometry, it was readily detected
by immunoprecipitation, suggesting that eosinophils contain
intracellular FcRI -chain, but express little or no FcRI on
their cell surface.
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RI -chain detected by
immunoprecipitation was not expressed at the cell surface, we performed
biotinylation of eosinophil cell surface proteins, followed by
immunoprecipitation of FcRI -chain. Although the FcRI
-chain was successfully immunoprecipitated, it was not detected by
streptavidin blotting for cell surface-biotinylated proteins (Fig. 3B). This approach also failed to detect -chain on the
surface of the eosinophils from atopic rhinitis subjects
(n = 3), one HES donor, and one late-phase BAL
eosinophil preparation (Fig. 3B and data not shown). In
contrast, surface biotinylation of basophils (as a positive control),
followed by immunoprecipitation of the FcRI -chain and
streptavidin blotting, revealed that this method is able to detect cell
surface expression of FcRI (Fig. 3B).
Detection of the FcRI - but not the ß-chains in
eosinophils
Because FcRI cell surface expression requires both FcRI -
and -chains, one possible explanation for the lack of cell surface
expression of FcRI, despite the presence of detectable FcRI
-chain protein, would be the lack of FcR -chain expression by
eosinophils. We found, however, that eosinophils do contain FcR
-chain detectable by immunoprecipitation in eosinophils from both
peripheral blood and BAL eosinophil preparations (Fig. 4A and data not shown).
Furthermore, cell surface biotinylation revealed that the -chain, in
contrast to the -chain, is present on the cell surface of
eosinophils (Fig. 4B). Thus, the failure of eosinophils to
express FcRI on the cell surface cannot be explained by a lack of
requisite FcR -chain expression. Since FcRI is a complex of three
distinct subunits, two of which we had detected in our eosinophil
lysates, we investigated the third subunit, FcRIß. Western blot
analysis of eosinophil lysates from allergic subjects failed to detect
FcRIß, even though it was easily detected in basophil lysates
(Fig. 4C and data not shown).
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RI -chain protein in the supernatant of
cultured eosinophils
Taking into account the undetectable surface expression, but clear
intracellular presence, of FcRI -chain protein, we hypothesized
that eosinophils may shed or secrete FcRI. To determine whether any
of the detected intracellular FcRI -chain is released
extracellularly, eosinophils were placed in culture for up to 8 days
with IL-5 (10 ng/ml). Culture supernatants were collected at various
time points by centrifugation and analyzed for the presence of FcRI
-chain by Western blotting. As shown in Fig. 5A, we detected a 50-kDa
protein in the supernatant that reacted with the FcRI
-chain-specific mAb 22E7 (n = 2) that was not
present in media alone. This protein was detected in supernatants as
early as 4 h of culture, and reached a plateau at approximately
48 h. It was also detected in supernatants of cells cultured for
24 h without IL-5 (data not shown). The viability of the cells at
this time point was 98%, suggesting that cell death and subsequent
release of intracellular -chain were not solely responsible for the
appearance of the -chain in the supernatants. We verified that the
protein detected in the supernatants was FcRI -chain by
immunoprecipitating FcRI -chain from eosinophil lysates and
eosinophil culture supernatants with another FcRI -chain-specific
mAb (15A5), followed by immunoblotting with the mAb 22E7
(n = 2) (Fig. 5B). This also allowed us to
compare the relative mass of FcRI in eosinophil lysates and
supernatants. The mass of FcRI -chain appearing in the
supernatants could not be accounted for based on changes in the
intracellular pools, providing further evidence that the protein found
in the lysate did not simply appear as a result of cell lysis. Taken
together, these results suggest that eosinophils accumulate and release
FcRI -chain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI was demonstrated
only on eosinophils from patients with marked eosinophilia (17).
Subsequent studies of atopic and nonatopic subjects have reported very
little, if any, surface expression of FcRI on peripheral blood
eosinophils (16, 34). This discrepancy raised the question of whether
the expression of FcRI on peripheral blood eosinophils is
donor specific or confined to diseases with eosinophilia. To
address this issue directly, we analyzed peripheral blood eosinophils
from normal donors and those with various conditions, ranging from mild
to severe eosinophilia. Immunofluorescence and flow cytometry revealed
no significant expression of FcRI, IgE, or CD23 on the surface of
eosinophils from 25 of our 26 donors. Only one eosinophil preparation,
from an asthmatic subject, showed a fold MFI greater than 1.5 (1.55)
with mAb 22E7; whether this slight increase corresponds to true
expression of FcRI on the surface of the cells or to variations
in the sensitivity of the assay is not possible to determine. It is
also of note that in this study we used the anti-FcRI mAb
22E7, which has been shown to be more sensitive in flow-cytometric
assays than mAb 15-1 (16) used in the Gounni et al. study on
hypereosinophilic subjects (17).
Several studies have demonstrated the presence of the subunit in
tissue-dwelling eosinophils (18, 34, 36, 37, 39, 40). In the study by
Terada et al., no surface expression of FcRI was detected on
peripheral blood eosinophils. However, double-labeling immunostaining
of nasal biopsies demonstrated the presence of FcRI+
eosinophils, suggesting that activation and migration of eosinophils to
sites of inflammation were necessary for expression of the receptor. In
this study, we have used late-phase BAL eosinophils, cells that have
been activated and undergone transmigration through the lung. There was
no significant surface expression of -chain, IgE, or CD23 in any of
the BAL samples we assayed, and thus no difference between surface
expression on peripheral blood eosinophils compared with BAL. These
results would appear to contradict the aforementioned studies (18, 34, 36, 37, 39, 40) and also to conflict with a recent report that
demonstrates increased levels of -chain mRNA and protein in
late-phase BAL eosinophils (38). In their study, 85–95% of
FcRI+ cells in cytospins from BAL fluid were
identified as eosinophils. A likely explanation for this apparent
discrepancy is the difference in the methods employed. In the
Rajakulasingam et al. study, as well as the other studies of tissue
eosinophils, investigators examined the presence of FcRI by
immunocytochemistry, a method that does not allow differentiation
between cell surface versus intracellular expression of the protein. In
contrast, we have utilized two independent methods, flow cytometry and
cell surface biotinylation, that were designed to detect FcRI
specifically expressed on the surface of cells. Furthermore, in
preliminary studies, immunofluorescence and flow-cytometric analysis of
eosinophils obtained by mechanical disruption of nasal polyps, another
rich source of tissue-dwelling eosinophils, failed to detect cell
surface FcR (unpublished observations).
Expression of FcRI on basophils has been shown to be regulated by
levels of IgE in vivo and in vitro (27, 32). In the latter study,
culture of basophils with IgE resulted in up-regulation of FcRI
in a linear time course during 2 wk of culture, as measured by
immunofluorescence and flow cytometry. The same IgE-mediated regulation
has been shown to occur in mouse and human mast cells (28, 35, 41).
Further regulation of FcRI expression is provided by IL-4, which has
been shown to up-regulate the receptor in cord blood-derived mast cells
and to increase mRNA levels in eosinophils (34, 42), and to induce
FcRII (CD23) on B cells (43). In the present study, surface
expression of FcRI or FcRII on eosinophils from a variety of
donors was not up-regulated following cell culture for up to 11 days
with IgE, IL-4, or a combination of both. These results suggest that
neither FcRI nor FcRII is induced on the surface of eosinophils
by IgE or IL-4, but leave open the possibility that IL-4 may regulate
the expression of FcR mRNA at the transcriptional level, as shown by
Terada et al. for FcRI (34). Although we were unable to detect
FcRI on the surface of eosinophils by flow cytometry,
immunocytochemistry studies have detected FcRI -chain protein in
eosinophils and mRNA by in situ hybridization or RT-PCR (37, 38, 39).
Indeed we have confirmed by immunoprecipitation analysis that
FcRI can be isolated from eosinophils. We, therefore, conclude
that eosinophils predominantly or exclusively express intracellular
FcRI protein. Whether this molecule is stored within eosinophil
granules or another intracellular location remains to be determined.
A potential reason for the lack of FcRI surface expression could
be the lack of FcR, because the expression of FcRI requires the
association of FcRI - with -chain. However, all of the
eosinophil preparations we tested expressed FcR on the cell surface,
confirming other studies showing mRNA for FcR in both peripheral
blood and tissue eosinophils (17, 37, 39). However, this is the first
report to detect FcR protein and its expression on the surface of
human eosinophils. FcR is not only essential for expression of
FcRI, but also FcRIII (CD16) (44), hence one possible explanation
for our findings is the presence of FcR in association with CD16 on
human eosinophils. Surface expression of CD16 has been reported
recently on approximately 6% of peripheral blood eosinophils from
atopic subjects (45). The report demonstrated that purification by
immunomagnetic separation of neutrophils, in a similar fashion as our
studies, successfully depletes eosinophils expressing surface CD16.
However, they detected a sizable pool of intracellular CD16 that was
capable of being mobilized to the surface of the cells by different
stimuli. Therefore, it is possible that the eosinophils we examined,
which also included one hypereosinophilic donor and a late-phase BAL
sample, expressed some CD16 on their surface.
Previous studies have also demonstrated mRNA for FcRI ß-chain in
eosinophils (17, 37, 39). However, we were unable to detect FcRIß
by Western blotting. Transfection studies have shown that FcR is
responsible for cell activation signals, whereas FcRß acts as an
amplifier of those signals (11). Furthermore, FcRI, composed of
and subunits and lacking the ß subunit, can be expressed on the
cell surface and can mediate similar signaling events as the
heterotrimeric receptor complex (46). It is therefore possible for
eosinophils to contain FcRI - and -chain protein without
expressing the ß-chain, as our study indicates.
In further studies, we examined the release of FcRI, based on the
premise that eosinophils possess a substantial intracellular pool of
FcRI, but do not express it on their surface. We detected a
protein in eosinophil culture supernatants that was similar in size
to FcRI found in eosinophil lysates. FcRI was detected
in culture supernatants by 4 h and increased over time. We ruled
out cell death and destruction as a sole source of FcRI in
culture supernatants, as cell viability remained at 98% for up to
72 h, by which point FcRI in the supernatant had already
reached a plateau. Furthermore, the relative amount of FcRI
immunoprecipitated from supernatants was comparable with that found in
whole cell lysates at the same time point. Flow cytometry performed at
24 and 48 h after culture, times at which protein was clearly
detectable in supernatants, again failed to reveal detectable surface
expression of FcRI -chain. These results would suggest that the
intracellular FcRI in eosinophils is secreted rather than cleaved
from the surface of the cell, but further studies are needed to clarify
this issue.
The existence of soluble FcR has been documented for IgG, IgA, IgD, and
IgE (reviewed in 47). Soluble receptors for IgE have been reported
as being derived from CD23, the low affinity IgE receptor, rather than
from the high affinity FcRI. Nonetheless, these soluble receptors
have been found to bind molecules other than IgE, and to regulate IgE
production, T cell and granulocyte maturation, and macrophage migration
(48). It is therefore possible that soluble FcRI -chain could act
to down-regulate cellular responses to IgE by binding free IgE and
decreasing its serum levels, as well as acting through non-IgE-mediated
pathways. Other reports have not only detected mRNA for FcRI ,
ß, and in human eosinophils, but also have seen an up-regulation
of FcRI mRNA expression in eosinophils. The enhanced mRNA
expression of FcRI was detected in eosinophils from late-phase
cutaneous reactions and late-phase BAL fluids (37, 38, 39). Based on those
studies, we would hypothesize that soluble FcRI has a biological
role in allergen-induced reactions. Moreover, the ability of mAb 15A5,
which binds to a peptide on the second domain of the FcRI
subunit and inhibits IgE binding (49), to detect -chain in the
supernatant of cultured eosinophils also indicates that this soluble
form of FcRI is capable of binding IgE. Previous reports have
shown that truncation of the transmembrane and cytoplasmic domains of
FcRI results in a protein secreted by Chinese hamster ovary cells
that retains its high affinity for IgE (50). Although from our studies
it is not possible to discern whether intracellular FcRI sequence
differs from FcRI expressed on the surface of the cells, future
investigations will concentrate on mRNA transcripts and protein
sequence to determine the molecular mechanism of FcRI release by
human eosinophils. Subsequent studies will also need to address whether
soluble FcRI is capable of binding IgE and to determine its
biological role.
In summary, eosinophils contain considerable amounts of intracellular
but not surface FcRI. Eosinophils do not express FcRIß or
FcRII (CD23) surface proteins, and we were unable to demonstrate
up-regulation of cell surface FcRI using IgE and IL-4, agents
that work for basophils and mast cells. The failure to express surface
FcRI could not be accounted for by a lack of FcR, as this was
easily detectable on the cell surface. The FcRI -chain is
released into the supernatant during culture with IL-5. Whether the
eosinophil transiently expresses FcRI on its surface, which is then
cleaved, or whether the FcRI -chain is directly secreted, is
still to be resolved. Moreover, the biological role of the soluble
FcRI is as yet unknown. We believe this study helps to clarify
discrepancies found in previous reports on the subject, and opens up
new avenues for investigation that should lead to a better
understanding of eosinophil and IgE receptor biology and the
pathogenesis of eosinophilic diseases.
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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: BAL, bronchoalveolar lavage; MFI, mean fluorescence intensity.
Received for publication November 10, 1998. Accepted for publication March 17, 1999.
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receptors (CD23) on normal human B lymphocytes. J. Exp. Med. 165:1459.-chain in tyrosine kinase activation via murine FcRIII. EMBO J. 11:2747.[Medline]
RIII (CD16) and its mobilization by chemoattractants in human eosinophils. J. Immunol. 161:2574.RI as a complex composed of FcRI- and FcRI-chains and can use this receptor for IgE-mediated allergen presentation. J. Immunol. 157:607.[Abstract]
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RI): analysis of functional domains of the -subunit with monoclonal antibodies. J. Biol. Chem. 266:11245. chain products from human, rat, and mouse high affinity receptor for immunoglobulin E. J. Biol. Chem. 266:2639.This article has been cited by other articles:
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