The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seminario, M.-C.
Right arrow Articles by Bochner, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seminario, M.-C.
Right arrow Articles by Bochner, B. S.
The Journal of Immunology, 1999, 162: 6893-6900.
Copyright © 1999 by The American Association of Immunologists

Intracellular Expression and Release of Fc{epsilon}RI{alpha} by Human Eosinophils1

Maria-Cristina Seminario, Sarbjit S. Saini, Donald W. MacGlashan, Jr. and Bruce S. Bochner2

Department of Medicine, Division of Clinical Immunology, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although Fc{epsilon}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, Fc{epsilon}RI, and Fc{epsilon}RII 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 Fc{epsilon}R on other cells) failed to induce any detectable surface Fc{epsilon}R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc{epsilon}RI{alpha} showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR {gamma}-chain, but no Fc{epsilon}RIß. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc{epsilon}RI{alpha}, although surface FcR{gamma} was easily detected. Since we were able to detect intracellular Fc{epsilon}RI{alpha}, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc{epsilon}RI{alpha} into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI{alpha} remains to be determined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eosinophils are proinflammatory leukocytes involved both in protection against parasites such as helminths (1) as well as in the pathogenesis of allergic diseases such as allergic rhinitis, asthma, and atopic dermatitis (2, 3). Eosinophils contain and release cationic proteins such as major basic protein, eosinophil peroxidase, and eosinophil cationic protein, which are potent cytotoxins that not only may cause tissue damage, but also serve to protect against helminthic infections (4). Eosinophils have also been shown to aggravate inflammation by their release of newly synthesized lipid mediators and cytokines (5).

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 (Fc{epsilon}RI) is a membrane glycoprotein composed of an {alpha} subunit, a ß subunit, and dimeric {gamma} subunits (9). The {alpha}-chain contains the extracellular IgE-binding domain (10), while the ß- and {gamma}-chains are felt to serve as intracellular signal transducers (11, 12). In human cells, expression of Fc{epsilon}RI requires the presence of the {alpha}- and {gamma}-chains, but not the ß-chain (13). Fc{epsilon}RI is primarily expressed by mast cells and basophils (14). Recent studies demonstrate, however, that Fc{epsilon}RI may also be expressed by epidermal Langerhans cells (15) and monocytes from some donors (16). Although cell surface expression of Fc{epsilon}RI 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 {alpha}-chain (18), cell surface levels of Fc{epsilon}RI, 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, Fc{epsilon}RII) 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Eosinophil analysis

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 IGo) (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).


View this table:
[in this window]
[in a new window]
  		Table I. Eosinophil blood donor characteristics

 
Basophil purification

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 Fc{epsilon}RI{alpha} protein on human basophils (27) and mouse mast cells (28).

Flow cytometry

Two different methods were used to examine expression of Fc{epsilon}R 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: Fc{epsilon}RI {alpha}-chain (22E7 and 15A5, kindly provided by Dr. J. Kochan, Hoffman-La Roche, Nutley, NJ), Fc{epsilon}RII (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 IGo) 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 Fc{epsilon}RI {alpha}-chain (22E7 or 15A5) bound to protein G-Sepharose, or with a FcR{gamma}-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 (Fc{epsilon}RI{alpha}), 15A5 (Fc{epsilon}RI{alpha}), 976 (Fc{epsilon}RIß-specific polyclonal rabbit antiserum, generously provided by Dr. J.-P. Kinet, Harvard University), or 934 (FcR{gamma}). 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 Fc{epsilon}RI{alpha} 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Surface expression of Fc{epsilon}R on purified eosinophils and eosinophils labeled in whole blood

Expression of Fc{epsilon}R 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 Fc{epsilon}RI{alpha} on eosinophils from normal donors or subjects with any of the eosinophilic conditions examined, as evidenced by staining with the mAb 22E7 (Fig. 1Go), which recognizes Fc{epsilon}RI 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 (Fc{epsilon}RII) 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 Fc{epsilon}RI{alpha} molecules could be discriminated from control antibody cytometric distributions ((33) and unpublished observations).



View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 1. Eosinophils from peripheral blood of patients from normal donors (n = 5) and those with diverse eosinophilic conditions (n = 21) or from late-phase BAL fluids (n = 5) express little or no Fc{epsilon}RI on their surface. Values are expressed as a ratio of MFI with the Fc{epsilon}RI {alpha}-chain Ab (22E7) divided by the MFI of the IgG control, with values for IgG control MFI of 0.48 ± 0.12. Bars represent means ± SEM, and p > 0.05 compared with a value of 1 for all 26 subjects. Experiments were performed on unpurified eosinophils labeled in whole blood (donors 1, 2, 12–16, and 22–26) or on purified eosinophils (donors 3–11 and 17–21), as described in Materials and Methods.

 
Culture of eosinophils with IgE and/or IL-4 does not up-regulate surface Fc{epsilon}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 Fc{epsilon}RI on basophils and mast cells and an up-regulation of Fc{epsilon}RI {alpha}-chain mRNA levels in human eosinophils (27, 28, 34, 35). Surface expression of Fc{epsilon}R and IgE was analyzed before culture and at 6 and 11 days of culture, with mAb 22E7 to the Fc{epsilon}RI {alpha}-chain, the FITC-conjugated polyclonal anti-IgE antiserum, and a mAb to CD23 (Fc{epsilon}RII). No significant increase in the levels of expression of any of the Fc{epsilon}R markers was seen for the different donors at any of the time points studied (Fig. 2Go and data not shown). We also performed immunofluorescence and flow cytometry at earlier time points, and again were unable to observe Fc{epsilon}RI{alpha} 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).



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. Culture of eosinophils with IL-5 for up to 11 days, with or without IgE, IL-4, or both, does not up-regulate surface expression of Fc{epsilon}RI. Shown are values for Fc{epsilon}RI{alpha}, expressed as a ratio of MFI with the Fc{epsilon}RI {alpha}-chain mAb 22E7 divided by the MFI of the IgG control. Eosinophils were purified from peripheral blood of: A, atopic donors (n = 8); B, hypereosinophilic donors (n = 5); C, atopic dermatitis donor (n = 1); or D, late-phase BAL fluid (n = 5). Data are presented as means ± SEM. Values for IgG control MFI were similar to those in Fig. 1Go.

 
Detection of Fc{epsilon}RI {alpha}-chain protein in eosinophil lysates

Although we did not detect significant cell surface expression of Fc{epsilon}RI, previous reports indicate that eosinophils contain Fc{epsilon}RI {alpha}-chain protein (as determined immunohistochemically) and mRNA-encoding Fc{epsilon}RI {alpha}-chain (17, 18, 34, 36, 37, 38, 39). {alpha}-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 Fc{epsilon}RI {alpha}-chain) was easily detectable in eosinophil lysates from a variety of donors (Fig. 3GoA, lane 1). In two of five experiments (including the one displayed in Fig. 3GoA, lane 1), a second fainter band of slightly higher relative m.w. was also detected. Fc{epsilon}RI {alpha}-chain was not immunoprecipitated with the control mAb and was also not detected in Jurkat T cell lysates (Fig. 3GoA). Furthermore, identical results were obtained when using a different immunoprecipitating mAb (15A5) reactive against another epitope of the {alpha}-chain (data not shown). In matching experiments, basophils expressed a protein of similar, although slightly lower, molecular mass (Fig. 3GoB), subtle differences likely due to differences in glycosylation. Thus, although Fc{epsilon}RI {alpha}-chain was not detected by flow cytometry, it was readily detected by immunoprecipitation, suggesting that eosinophils contain intracellular Fc{epsilon}RI {alpha}-chain, but express little or no Fc{epsilon}RI on their cell surface.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3. Intracellular but not surface expression of Fc{epsilon}RI{alpha} by human eosinophils. Shown are Western blots using eosinophils isolated from donor 4 representative of five to six separate experiments with similar results using peripheral blood or BAL eosinophils from donors 4, 5, 6, 12, 20, and 21. A, Detection of Fc{epsilon}RI{alpha} immunoprecipitated from eosinophil lysates with mAb 22E7 (lane 1); immunoprecipitation with an isotype-matched IgG control (lane 2); {alpha}-chain immunoprecipitated from Jurkat cells with mAb 22E7 (lane 3), used as negative controls. The membrane was immunoblotted with mAb 15A5 (Fc{epsilon}RI {alpha}-chain). B, The upper panel shows the presence of Fc{epsilon}RI {alpha}-chain immunoprecipitated from surface-biotinylated basophils (lane 1) or eosinophils (lane 2) with mAb 22E7, followed by immunoblotting with mAb 22E7; the lower panel shows surface expression of Fc{epsilon}RI{alpha} on basophils, but not eosinophils after blotting with HRP-conjugated streptavidin. IP, immunoprecipitation; IB, immunoblotting.

 
To confirm further that the Fc{epsilon}RI {alpha}-chain detected by immunoprecipitation was not expressed at the cell surface, we performed biotinylation of eosinophil cell surface proteins, followed by immunoprecipitation of Fc{epsilon}RI {alpha}-chain. Although the Fc{epsilon}RI {alpha}-chain was successfully immunoprecipitated, it was not detected by streptavidin blotting for cell surface-biotinylated proteins (Fig. 3GoB). This approach also failed to detect {alpha}-chain on the surface of the eosinophils from atopic rhinitis subjects (n = 3), one HES donor, and one late-phase BAL eosinophil preparation (Fig. 3GoB and data not shown). In contrast, surface biotinylation of basophils (as a positive control), followed by immunoprecipitation of the Fc{epsilon}RI {alpha}-chain and streptavidin blotting, revealed that this method is able to detect cell surface expression of Fc{epsilon}RI (Fig. 3GoB).

Detection of the Fc{epsilon}RI {gamma}- but not the ß-chains in eosinophils

Because Fc{epsilon}RI cell surface expression requires both Fc{epsilon}RI {alpha}- and {gamma}-chains, one possible explanation for the lack of cell surface expression of Fc{epsilon}RI, despite the presence of detectable Fc{epsilon}RI {alpha}-chain protein, would be the lack of FcR {gamma}-chain expression by eosinophils. We found, however, that eosinophils do contain FcR {gamma}-chain detectable by immunoprecipitation in eosinophils from both peripheral blood and BAL eosinophil preparations (Fig. 4GoA and data not shown). Furthermore, cell surface biotinylation revealed that the {gamma}-chain, in contrast to the {alpha}-chain, is present on the cell surface of eosinophils (Fig. 4GoB). Thus, the failure of eosinophils to express Fc{epsilon}RI on the cell surface cannot be explained by a lack of requisite FcR {gamma}-chain expression. Since Fc{epsilon}RI is a complex of three distinct subunits, two of which we had detected in our eosinophil lysates, we investigated the third subunit, Fc{epsilon}RIß. Western blot analysis of eosinophil lysates from allergic subjects failed to detect Fc{epsilon}RIß, even though it was easily detected in basophil lysates (Fig. 4GoC and data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 4. Western blot analysis of FcR{gamma} (A and B) and Fc{epsilon}RIß protein (C) in eosinophils. A, lane 1, Isotype-matched IgG was used as negative control for immunoprecipitation; lanes 2 and 3, clearly distinguishable FcR{gamma} protein immunoprecipitated from eosinophil or basophil lysates with FcR{gamma}-specific polyclonal rabbit antiserum (934); lane 4, FcR{gamma} protein immunoprecipitated from Jurkat cells as negative controls. The membrane was immunoblotted with antiserum 934 to FcR{gamma}. Data are representative of four separate experiments (donors 4, 5, 14, and 19) with similar results. B, Detectable FcR{gamma} protein immunoprecipitated from surface-biotinylated eosinophils with FcR{gamma}-specific polyclonal rabbit antiserum (934) immunoblotted with either antisera 934 (lane 1) or streptavidin (lane 2). Lane 3 shows the control IgG immunoprecipitation. IP, immunoprecipitation; IB, immunoblotting. Data are representative of three separate experiments (donors 5, 12, and 19) with similar results. C, Western blot analysis using Fc{epsilon}RIß-specific polyclonal rabbit antiserum (976), showing the presence of Fc{epsilon}RI ß-chain in basophils (lane 2), but not in eosinophils (lane 1). Data are representative of three separate experiments (donors 4, 6, and 12) with similar results.

 
Appearance of Fc{epsilon}RI {alpha}-chain protein in the supernatant of cultured eosinophils

Taking into account the undetectable surface expression, but clear intracellular presence, of Fc{epsilon}RI {alpha}-chain protein, we hypothesized that eosinophils may shed or secrete Fc{epsilon}RI. To determine whether any of the detected intracellular Fc{epsilon}RI {alpha}-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 Fc{epsilon}RI {alpha}-chain by Western blotting. As shown in Fig. 5GoA, we detected a 50-kDa protein in the supernatant that reacted with the Fc{epsilon}RI {alpha}-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 {alpha}-chain were not solely responsible for the appearance of the {alpha}-chain in the supernatants. We verified that the protein detected in the supernatants was Fc{epsilon}RI {alpha}-chain by immunoprecipitating Fc{epsilon}RI {alpha}-chain from eosinophil lysates and eosinophil culture supernatants with another Fc{epsilon}RI {alpha}-chain-specific mAb (15A5), followed by immunoblotting with the mAb 22E7 (n = 2) (Fig. 5GoB). This also allowed us to compare the relative mass of Fc{epsilon}RI{alpha} in eosinophil lysates and supernatants. The mass of Fc{epsilon}RI {alpha}-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 Fc{epsilon}RI {alpha}-chain.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 5. Presence of Fc{epsilon}RI{alpha} in eosinophil culture supernatants. A, Representative Western blot of two separate experiments (donors 4 and 12) with similar results showing the presence of a protein that reacted with the Fc{epsilon}RI{alpha} mAb 22E7 in eosinophil culture supernatants at various time points after culture with IL-5 (10 ng/ml). B, Confirmation that the protein detected in the supernatants is Fc{epsilon}RI {alpha}-chain, as evidenced by Fc{epsilon}RI{alpha} immunoprecipitation from 1) eosinophil lysates after purification, 2) eosinophil lysates after 24 h in culture, and 3) eosinophil culture supernatants, with another Fc{epsilon}RI {alpha}-chain-specific mAb (15A5), followed by immunoblotting with the mAb 22E7 (donors 4 and 12). The relative amount of Fc{epsilon}RI{alpha} detected in eosinophils and their culture supernatant indicates that the soluble protein is not solely derived from eosinophil lysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We undertook this study in an attempt to resolve the controversies regarding the expression of IgE receptors on human eosinophils. For example, significant surface expression of Fc{epsilon}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 Fc{epsilon}RI on peripheral blood eosinophils (16, 34). This discrepancy raised the question of whether the expression of Fc{epsilon}RI 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 Fc{epsilon}RI{alpha}, 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 Fc{epsilon}RI{alpha} 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-Fc{epsilon}RI{alpha} 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 {alpha} subunit in tissue-dwelling eosinophils (18, 34, 36, 37, 39, 40). In the study by Terada et al., no surface expression of Fc{epsilon}RI{alpha} was detected on peripheral blood eosinophils. However, double-labeling immunostaining of nasal biopsies demonstrated the presence of Fc{epsilon}RI{alpha}+ 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 {alpha}-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 {alpha}-chain mRNA and protein in late-phase BAL eosinophils (38). In their study, 85–95% of Fc{epsilon}RI{alpha}+ 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 Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI 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 Fc{epsilon}R (unpublished observations).

Expression of Fc{epsilon}RI 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 Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI 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 Fc{epsilon}RII (CD23) on B cells (43). In the present study, surface expression of Fc{epsilon}RI{alpha} or Fc{epsilon}RII 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 Fc{epsilon}RI nor Fc{epsilon}RII 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 Fc{epsilon}R mRNA at the transcriptional level, as shown by Terada et al. for Fc{epsilon}RI{alpha} (34). Although we were unable to detect Fc{epsilon}RI on the surface of eosinophils by flow cytometry, immunocytochemistry studies have detected Fc{epsilon}RI {alpha}-chain protein in eosinophils and mRNA by in situ hybridization or RT-PCR (37, 38, 39). Indeed we have confirmed by immunoprecipitation analysis that Fc{epsilon}RI{alpha} can be isolated from eosinophils. We, therefore, conclude that eosinophils predominantly or exclusively express intracellular Fc{epsilon}RI{alpha} protein. Whether this molecule is stored within eosinophil granules or another intracellular location remains to be determined.

A potential reason for the lack of Fc{epsilon}RI{alpha} surface expression could be the lack of FcR{gamma}, because the expression of Fc{epsilon}RI requires the association of Fc{epsilon}RI {alpha}- with {gamma}-chain. However, all of the eosinophil preparations we tested expressed FcR{gamma} on the cell surface, confirming other studies showing mRNA for FcR{gamma} in both peripheral blood and tissue eosinophils (17, 37, 39). However, this is the first report to detect FcR{gamma} protein and its expression on the surface of human eosinophils. FcR{gamma} is not only essential for expression of Fc{epsilon}RI, but also Fc{gamma}RIII (CD16) (44), hence one possible explanation for our findings is the presence of FcR{gamma} 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 Fc{epsilon}RI ß-chain in eosinophils (17, 37, 39). However, we were unable to detect Fc{epsilon}RIß by Western blotting. Transfection studies have shown that FcR{gamma} is responsible for cell activation signals, whereas FcRß acts as an amplifier of those signals (11). Furthermore, Fc{epsilon}RI, composed of {alpha} and {gamma} 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 Fc{epsilon}RI {alpha}- and {gamma}-chain protein without expressing the ß-chain, as our study indicates.

In further studies, we examined the release of Fc{epsilon}RI{alpha}, based on the premise that eosinophils possess a substantial intracellular pool of Fc{epsilon}RI{alpha}, but do not express it on their surface. We detected a protein in eosinophil culture supernatants that was similar in size to Fc{epsilon}RI{alpha} found in eosinophil lysates. Fc{epsilon}RI{alpha} was detected in culture supernatants by 4 h and increased over time. We ruled out cell death and destruction as a sole source of Fc{epsilon}RI{alpha} in culture supernatants, as cell viability remained at 98% for up to 72 h, by which point Fc{epsilon}RI{alpha} in the supernatant had already reached a plateau. Furthermore, the relative amount of Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI {alpha}-chain. These results would suggest that the intracellular Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI. 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 Fc{epsilon}RI {alpha}-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 Fc{epsilon}RI {alpha}, ß, and {gamma} in human eosinophils, but also have seen an up-regulation of Fc{epsilon}RI{alpha} mRNA expression in eosinophils. The enhanced mRNA expression of Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI {alpha} subunit and inhibits IgE binding (49), to detect {alpha}-chain in the supernatant of cultured eosinophils also indicates that this soluble form of Fc{epsilon}RI{alpha} is capable of binding IgE. Previous reports have shown that truncation of the transmembrane and cytoplasmic domains of Fc{epsilon}RI{alpha} 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 Fc{epsilon}RI{alpha} sequence differs from Fc{epsilon}RI{alpha} expressed on the surface of the cells, future investigations will concentrate on mRNA transcripts and protein sequence to determine the molecular mechanism of Fc{epsilon}RI{alpha} release by human eosinophils. Subsequent studies will also need to address whether soluble Fc{epsilon}RI{alpha} is capable of binding IgE and to determine its biological role.

In summary, eosinophils contain considerable amounts of intracellular but not surface Fc{epsilon}RI{alpha}. Eosinophils do not express Fc{epsilon}RIß or Fc{epsilon}RII (CD23) surface proteins, and we were unable to demonstrate up-regulation of cell surface Fc{epsilon}RI{alpha} using IgE and IL-4, agents that work for basophils and mast cells. The failure to express surface Fc{epsilon}RI could not be accounted for by a lack of FcR{gamma}, as this was easily detectable on the cell surface. The Fc{epsilon}RI {alpha}-chain is released into the supernatant during culture with IL-5. Whether the eosinophil transiently expresses Fc{epsilon}RI on its surface, which is then cleaved, or whether the Fc{epsilon}RI {alpha}-chain is directly secreted, is still to be resolved. Moreover, the biological role of the soluble Fc{epsilon}RI{alpha} 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.


   Acknowledgments
 
We thank Drs. Ishizaka, Kochan, and Kinet for providing valuable reagents; Dr. Hirohito Kita for his critical review of the manuscript; and Bonnie Hebden for assistance in the preparation of the manuscript.


   Footnotes
 
1 This work was supported by Grants HL49545 and AI33372 from the National Institutes of Health and an Underrepresented Minority Investigator in Asthma and Allergy Award to M.-C.S from the National Institutes of Health and the American Academy of Allergy, Asthma and Immunology. B.S.B. was also supported in part by a Developing Investigator Award from the Burroughs Wellcome Fund and a grant from the Office of Naval Research, awarded through the Asthma and Allergy Foundation of America. Back

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: Back

3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; MFI, mean fluorescence intensity. Back

Received for publication November 10, 1998. Accepted for publication March 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wardlaw, A. J., R. Moqbel. 1992. The eosinophil in allergic and helminth-related inflammatory responses. R. Moqbel, ed. Allergy and Immunity to Helminths: Common Mechanisms or Divergent Pathways? 154. Taylor & Francis, Bristol.
  2. Butterfield, J. H., K. M. Leiferman, G. J. Gleich. 1995. Eosinophil-associated diseases. M. M. Frank, and K. F. Austen, and H. N. Claman, and E. R. Unanue, eds. Samter’s Immunological Diseases 5th Ed.501. Little, Brown, and Company, Boston.
  3. Seminario, M. C., G. J. Gleich. 1994. The role of eosinophils in the pathogenesis of asthma. Curr. Opin. Immunol. 6:860.[Medline]
  4. Seminario, M.-C., C. Adolphson, G. J. Gleich. 1998. Eosinophils: granule proteins. W. W. Busse, and S. T. Holgate, eds. Inflammatory Mechanisms in Asthma 157. Marcel Dekker, New York.
  5. Gleich, G., C. Adolphson, K. Leiferman. 1993. The biology of the eosinophilic leukocyte. Annu. Rev. Med. 44:85.[Medline]
  6. Hagan, P., U. Blumenthal, D. Dunn, A. Simpson, H. Wilkins. 1991. Human IgE, IgG4 and resistance to reinfection with Schistosoma haematobium. Nature 349:243.[Medline]
  7. Dunne, D., A. Butterworth, A. Fulford, H. Kariuki, J. Langley, J. Ouma, A. Capron, R. Pierce, R. Sturrock. 1992. Immunity after treatment of human schistosomiasis: association between IgE antibodies to adult worm antigens and resistance to reinfection. Eur. J. Immunol. 22:1483.[Medline]
  8. Pritchard, D., E. Walsh. 1995. The specificity of the human IgE response to Necator americanus. Parasite Immunol. 17:605.[Medline]
  9. Ravetch, J.. 1994. Fc receptors: rubor redux. Cell 78:553.[Medline]
  10. Blank, U., C. Ra, L. Miller, K. White, H. Metzger, J. Kinet. 1989. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 337:187.[Medline]
  11. Lin, S. Q., C. Cicala, A. M. Scharenberg, J. P. Kinet. 1996. The Fc{epsilon}RIß subunit functions as an amplifier of Fc{epsilon}RI{gamma}-mediated cell activation signals. Cell 85:985.[Medline]
  12. Paolini, R., R. Numerof, J. P. Kinet. 1992. Phosphorylation/dephosphorylation of high-affinity IgE receptors: a mechanism for coupling/uncoupling a large signaling complex. Proc. Natl. Acad. Sci. USA 89:10733.[Abstract/Free Full Text]
  13. Miller, L., U. Blank, H. Metzger, J. Kinet. 1989. Expression of high-affinity binding of human immunoglobulin E by transfected cells. Science 244:334.[Abstract/Free Full Text]
  14. Ravetch, J. V., J. P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[Medline]
  15. Wang, B., A. Rieger, O. Kilgus, K. Ochiai, D. Maurer, D. Fodinger, J.-P. Kinet, G. Sting. 1992. Epidermal Langerhans cells from normal human skin bind monomeric IgE via Fc{epsilon}RI. J. Exp. Med. 175:1353.[Abstract/Free Full Text]
  16. Sihra, B. S., O. M. Kon, J. A. Grant, A. B. Kay. 1997. Expression of high-affinity IgE receptors (Fc{epsilon}RI) on peripheral blood basophils, monocytes, and eosinophils in atopic and nonatopic subjects: relationship to total serum IgE concentrations. J. Allergy Clin. Immunol. 99:699.[Medline]
  17. Gounni, A. S., B. Lamkhioued, K. Ochial, Y. Tanaka, E. Delaporte, A. Capron, J. P. Kinet, M. Capron. 1994. High-affinity IgE receptor on eosinophils is involved in defense against parasites. Nature 367:183.[Medline]
  18. Rajakulasingam, K., S. R. Durham, F. O’Brien, M. Humbert, L. T. Barata, L. Reece, A. B. Kay, J. A. Grant. 1997. Enhanced expression of high-affinity IgE receptor (Fc{epsilon}RI) {alpha} chain in human allergen-induced rhinitis with co-localization to mast cells, macrophages, eosinophils, and dendritic cells. J. Allergy Clin. Immunol. 100:78.[Medline]
  19. Capron, M., M. J. Truong, D. Aldebert, V. Gruart, M. Suemura, G. Delespesse, B. Tourvieille, A. Capron. 1991. Heterogeneous expression of CD23 epitopes by eosinophils from patients: relationships with IgE-mediated functions. Eur. J. Immunol. 21:2423.[Medline]
  20. Hansel, T. T., I. J. M. D. Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105.[Medline]
  21. Liu, M. C., W. C. Hubbard, D. Proud, B. Stealey, S. Galli, A. Kagey-Sobotka, E. R. Bleecker, L. M. Lichtenstein. 1991. Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in asthmatics: cellular, mediator, and permeability changes. Am. Rev. Respir. Dis. 144:51.[Medline]
  22. Kroegel, C., M. C. Liu, W. M. Hubbard, L. M. Lichtenstein, B. S. Bochner. 1994. Blood and bronchoalveolar eosinophils in allergic subjects following segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production. J. Allergy Clin. Immunol. 93:725.[Medline]
  23. Georas, S. N., M. C. Liu, W. Newman, W. D. Beall, B. A. Stealey, B. S. Bochner. 1992. Altered adhesion molecule expression and endothelial activation accompany the recruitment of human granulocytes to the lung following segmental antigen challenge. Am. J. Respir. Cell Mol. Biol. 7:261.
  24. Matsumoto, K., J. Appiah-Pippim, R. P. Schleimer, C. A. Bickel, L. A. Beck, B. S. Bochner. 1998. CD44 and CD69 represent different types of cell surface activation markers for human eosinophils. Am. J. Respir. Cell Mol. Biol. 18:860.[Abstract/Free Full Text]
  25. Warner, J. A., Jr D. W. MacGlashan. 1988. Persistence of early crosslink-dependent signal transduction events in human basophils after desensitization. Immunol. Lett. 18:129.[Medline]
  26. Matsumoto, K., R. P. Schleimer, H. Saito, Y. Iikura, B. S. Bochner. 1995. Induction of apoptosis in human eosinophils by anti-fas antibody treatment in vitro. Blood 86:1437.[Abstract/Free Full Text]
  27. Jr MacGlashan, D. W., J. McKenzie White, K. Chichester, B. S. Bochner, F. M. Davis, J. T. Schroeder, L. M. Lichtenstein. 1998. In vitro regulation of Fc{epsilon}RI{alpha} expression on human basophils by IgE antibody. Blood 91:1633.[Abstract/Free Full Text]
  28. Yamaguchi, M., C. S. Lantz, H. C. Oettgen, I. M. Katona, T. Fleming, I. Miyajima, J.-P. Kinet, S. J. Galli. 1997. IgE enhances mouse mast cell Fc{epsilon}RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions. J. Exp. Med. 185:663.[Abstract/Free Full Text]
  29. Bochner, B. S., A. A. McKelvey, R. P. Schleimer, J. E. K. Hildreth, Jr D. W. MacGlashan. 1989. Flow cytometric methods for analysis of human basophil surface antigens and viability. J. Immunol. Methods 125:265.[Medline]
  30. Boulet, J. P., A. Cartier, N. C. Thomson, R. S. Roberts, J. Dolovich, F. E. Hargreave. 1983. Asthma and increases in nonallergic bronchial responsiveness from seasonal pollen exposure. J. Allergy Clin. Immunol. 71:399.[Medline]
  31. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  32. Jr MacGlashan, D. W., B. S. Bochner, D. C. Adelman, P. M. Jardieu, A. Togias, J. McKenzie-White, S. A. Sterbinsky, R. G. Hamilton, L. M. Lichtenstein. 1997. Down-regulation of Fc{epsilon}RI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody. J. Immunol. 158:1438.[Abstract]
  33. Wantke, F., D. W. MacGlashan Jr., J. M. Langdon, and S. M. MacDonald. 1999. The recombinant histamine releasing factor rHRF: functional evidence that rHRF does not bind to the IgE molecule. J. Allergy Clin. Immunol. In press.
  34. Terada, N., A. Konno, Y. Terada, S. Fukuda, T. Yamashita, T. Abe, H. Shimada, K. Ishida, K. Yoshimura, Y. Tanaka, C. Ra, K. Ishikawa, K. Togawa. 1995. IL-4 up-regulates Fc{epsilon}RI {alpha}-chain mRNA in eosinophils. J. Allergy Clin. Immunol. 96:1161.[Medline]
  35. Hsu, C., Jr D. MacGlashan. 1996. IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells. Immunol. Lett. 52:129.[Medline]
  36. Humbert, M., J. A. Grant, L. Tabordabarata, S. R. Durham, R. Pfister, G. Menz, J. Barkans, S. Ying, A. B. Kay. 1996. High-affinity IgE receptor (Fc{epsilon}RI)-bearing cells in bronchial biopsies from atopic and nonatopic asthma. Am. J. Resp. Crit. Care Med. 153:1931.[Abstract]
  37. Barata, L. T., S. Ying, J. A. Grant, M. Humbert, J. Barkans, Q. Meng, S. R. Durham, A. B. Kay. 1997. Allergen-induced recruitment of Fc{epsilon}RI+ eosinophils in human atopic skin. Eur. J. Immunol. 27:1236.[Medline]
  38. Rajakulasingam, K., S. Till, S. Ying, M. Humbert, J. Barkans, M. Sullivan, Q. Meng, C. J. Corrigan, J. Bungre, J. A. Grant, A. B. Kay, S. R. Durham. 1998. Increased expression of high affinity IgE (Fc{epsilon}RI) receptor-{alpha} chain mRNA and protein-bearing eosinophils in human allergen-induced atopic asthma. Am. J. Respir. Crit. Care Med. 158:233.[Abstract/Free Full Text]
  39. Ying, S., L. T. Barata, Q. Meng, J. A. Grant, J. Barkans, S. R. Durham, A. B. Kay. 1998. High-affinity immunoglobulin E receptor (Fc{epsilon}RI)-bearing eosinophils, mast cells, macrophages and Langerhans’ cells in allergen-induced late-phase cutaneous reactions in atopic subjects. Immunology 93:281.[Medline]
  40. Gounni, A. S., B. Lamkhioued, E. Delaporte, A. Dubost, J. P. Kinet, A. Capron, M. Capron. 1994. The high-affinity IgE receptor on eosinophils: from allergy to parasites or from parasites to allergy?. J. Allergy Clin. Immunol. 94:1214.[Medline]
  41. Yamaguchi, M., C. Z. Lantz, H. C. Oettgen, I. M. Katona, T. Fleming, K. Yano, I. Miyajima, J. P. Kinet, S. J. Galli. 1997. IgE enhances mouse and human mast cell Fc{epsilon}RI expression, which in turn enhances IgE-dependent mast cell mediator release. J. Allergy Clin. Immunol. 99:S104.
  42. Toru, H., C. Ra, S. Nonoyama, K. Suzuki, J. Yata, T. Nakahata. 1997. Induction of high affinity IgE receptor on human mast cells by IL-4. J. Allergy Clin. Immunol. 99:S103.
  43. DeFrance, T., J. P. Aubry, F. Rousset, B. Vanbervliet, J. Y. Bonnefoy, N. Arai, Y. Takebe, T. Yokota, F. Lee, K. Arai, J. de Vries, J. Banchereau. 1987. Human recombinant interleukin 4 induces Fc{epsilon} receptors (CD23) on normal human B lymphocytes. J. Exp. Med. 165:1459.[Abstract/Free Full Text]
  44. Bonnerot, C., S. Amigorena, D. Choquet, R. Pavlovich, V. Choukroun, W. Fridman. 1992. Role of associated {gamma}-chain in tyrosine kinase activation via murine Fc{gamma}RIII. EMBO J. 11:2747.[Medline]
  45. Zhu, X. D., K. J. Hamann, N. M. Munoz, N. Rubio, D. Mayer, A. Hernrreiter, A. R. Leff. 1998. Intracellular expression of Fc{gamma}RIII (CD16) and its mobilization by chemoattractants in human eosinophils. J. Immunol. 161:2574.[Abstract/Free Full Text]
  46. Maurer, D., E. Fiebiger, C. Ebner, B. Reininger, G. F. Fischer, S. Wichlas, M. H. Jouvin, M. Schmittegenolf, D. Kraft, J. P. Kinet, G. Stingl. 1996. Peripheral blood dendritic cells express Fc{epsilon}RI as a complex composed of Fc{epsilon}RI{alpha}- and Fc{epsilon}RI{gamma}-chains and can use this receptor for IgE-mediated allergen presentation. J. Immunol. 157:607.[Abstract]
  47. Fridman, W., J. Teillaud, B. C. C. Teillaud, A. Astier, E. Tartour, J. Galon, C. Mathiot, C. Sautes. 1993. Soluble Fc{gamma} receptors. J. Leukocyte Biol. 54:504.[Abstract]
  48. Delespesse, G., M. Sarfati, C. Wu, S. Fournier, M. Letellier. 1992. The low-affinity receptor for IgE. Immunol. Rev. 125:77.[Medline]
  49. Riske, F., J. Hakimi, M. Mallamaci, M. Griffin, B. Pilson, N. Tobkes, P. Lin, W. Danho, J. Kochan, R. Chizzonite. 1991. High affinity human IgE receptor (Fc{epsilon}RI): analysis of functional domains of the {alpha}-subunit with monoclonal antibodies. J. Biol. Chem. 266:11245.[Abstract/Free Full Text]
  50. Blank, U., C. Ra, J. Kinet. 1991. Characterization of truncated {alpha} chain products from human, rat, and mouse high affinity receptor for immunoglobulin E. J. Biol. Chem. 266:2639.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Ganguly, C. Grodzki, D. Sugden, M. Moller, S. Odom, P. Gaildrat, I. Gery, R. P. Siraganian, J. Rivera, and D. C. Klein
Neural Adrenergic/Cyclic AMP Regulation of the Immunoglobulin E Receptor {alpha}-Subunit Expression in the Mammalian Pinealocyte: A NEUROENDOCRINE/IMMUNE RESPONSE LINK?
J. Biol. Chem., November 9, 2007; 282(45): 32758 - 32764.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. M. Cauvi, X. Tian, K. von Loehneysen, and M. W. Robertson
Transport of the IgE Receptor {alpha}-Chain Is Controlled by a Multicomponent Intracellular Retention Signal
J. Biol. Chem., April 14, 2006; 281(15): 10448 - 10460.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
L. M. Ganley-Leal, P. N. Mwinzi, C. B. Cetre-Sossah, J. Andove, A. W. Hightower, D. M. S. Karanja, D. G. Colley, and W. E. Secor
Correlation between Eosinophils and Protection against Reinfection with Schistosoma mansoni and the Effect of Human Immunodeficiency Virus Type 1 Coinfection in Humans
Infect. Immun., April 1, 2006; 74(4): 2169 - 2176.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
D. Ferrari, A. la Sala, E. Panther, J. Norgauer, F. Di Virgilio, and M. Idzko
Activation of human eosinophils via P2 receptors: novel findings and future perspectives
J. Leukoc. Biol., January 1, 2006; 79(1): 7 - 15.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. T. Schroeder, A. P. Bieneman, H. Xiao, K. L. Chichester, K. Vasagar, S. Saini, and M. C. Liu
TLR9- and Fc{epsilon}RI-Mediated Responses Oppose One Another in Plasmacytoid Dendritic Cells by Down-Regulating Receptor Expression
J. Immunol., November 1, 2005; 175(9): 5724 - 5731.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Inoue, Y. Matsuwaki, S.-H. Shin, J. U. Ponikau, and H. Kita
Nonpathogenic, Environmental Fungi Induce Activation and Degranulation of Hu