Although FcεR 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, FcεRI, and FcεRII 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 FcεR on other cells) failed to induce any detectable surface FcεR. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for FcεRIα showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR γ-chain, but no FcεRIβ. Surface biotinylation followed by immunoprecipitation again failed to detect surface FcεRIα, although surface FcRγ was easily detected. Since we were able to detect intracellular FcεRIα, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of FcεRIα into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of FcεRIα 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 FcεRIα remains to be determined.

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

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).

Table I.

Eosinophil blood donor characteristics

SubjectAgeSexDiagnosis% EosTotal IgE (ng/ml)
50 Normala 21 
34 Normala <1 52 
33 Allergic rhinitis 90 
39 Allergic rhinitis 164 
33 Allergic rhinitis 346 
31 Allergic rhinitis 267 
23 Asthma NDb 1,115 
39 Asthma 2,469 
44 Asthma ND 145 
10 26 Asthma 451 
11 48 Asthmaa ND ND 
12 25 Allergic rhinitis and asthma 11 1,613 
13 27 Allergic rhinitis and asthma 268 
14 40 Allergic rhinitis and asthma ND 
15 58 Allergic rhinitis and asthma 12 ND 
16 36 Atopic dermatitis 8,832 
17 40 Eosinophilic gastroenteritis 20 534 
18 28 Eosinophilic pneumonia, nasal polyposis 12 123 
19 63 Episodic eosinophilia with angioedema 29 29 
20 66 Hypereosinophilic syndrome 26 30,840 
21 65 Hypereosinophilic syndromea 41 ND 
22 69 IgE myeloma ND 4.3× 106 
23 59 Lymphatic filariasis 16 792 
24 31 Normala ND 
25 39 Normala ND 
26 27 Normala <1 ND 
SubjectAgeSexDiagnosis% EosTotal IgE (ng/ml)
50 Normala 21 
34 Normala <1 52 
33 Allergic rhinitis 90 
39 Allergic rhinitis 164 
33 Allergic rhinitis 346 
31 Allergic rhinitis 267 
23 Asthma NDb 1,115 
39 Asthma 2,469 
44 Asthma ND 145 
10 26 Asthma 451 
11 48 Asthmaa ND ND 
12 25 Allergic rhinitis and asthma 11 1,613 
13 27 Allergic rhinitis and asthma 268 
14 40 Allergic rhinitis and asthma ND 
15 58 Allergic rhinitis and asthma 12 ND 
16 36 Atopic dermatitis 8,832 
17 40 Eosinophilic gastroenteritis 20 534 
18 28 Eosinophilic pneumonia, nasal polyposis 12 123 
19 63 Episodic eosinophilia with angioedema 29 29 
20 66 Hypereosinophilic syndrome 26 30,840 
21 65 Hypereosinophilic syndromea 41 ND 
22 69 IgE myeloma ND 4.3× 106 
23 59 Lymphatic filariasis 16 792 
24 31 Normala ND 
25 39 Normala ND 
26 27 Normala <1 ND 
a

Routine allergy skin testing was entirely negative.

b

ND, not determined.

Peripheral blood basophils were isolated by elutriation and Percoll density gradients of leukopheresis packs to >60% purity (25).

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 FcεRIα protein on human basophils (27) and mouse mast cells (28).

Two different methods were used to examine expression of FcεR 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: FcεRI α-chain (22E7 and 15A5, kindly provided by Dr. J. Kochan, Hoffman-La Roche, Nutley, NJ), FcεRII (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.

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 × g, 5 min, 4°C). For Western blot analysis of whole cell extracts, lysates from 2 × 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 × 106 cells were incubated for 2 h at 4°C with mAbs to the FcεRI α-chain (22E7 or 15A5) bound to protein G-Sepharose, or with a FcRγ-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 (FcεRIα), 15A5 (FcεRIα), 976 (FcεRIβ-specific polyclonal rabbit antiserum, generously provided by Dr. J.-P. Kinet, Harvard University), or 934 (FcRγ). 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).

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.

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 × 106 eosinophils were lysed and FcεRIα immunoprecipitated immediately after purification. A separate aliquot of 3 × 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.

All data are expressed as mean ± SEM. Differences between groups were assessed by ANOVA. Statistical significance was reached at p < 0.05.

Expression of FcεR 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 FcεRIα 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 FcεRI 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 (FcεRII) 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 FcεRIα molecules could be discriminated from control antibody cytometric distributions ((33) and unpublished observations).

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 FcεRI on their surface. Values are expressed as a ratio of MFI with the FcεRI α-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.

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 FcεRI on their surface. Values are expressed as a ratio of MFI with the FcεRI α-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.

Close modal

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 FcεRI on basophils and mast cells and an up-regulation of FcεRI α-chain mRNA levels in human eosinophils (27, 28, 34, 35). Surface expression of FcεR and IgE was analyzed before culture and at 6 and 11 days of culture, with mAb 22E7 to the FcεRI α-chain, the FITC-conjugated polyclonal anti-IgE antiserum, and a mAb to CD23 (FcεRII). No significant increase in the levels of expression of any of the FcεR 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 FcεRIα 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).

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 FcεRI. Shown are values for FcεRIα, expressed as a ratio of MFI with the FcεRI α-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. 1.

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 FcεRI. Shown are values for FcεRIα, expressed as a ratio of MFI with the FcεRI α-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. 1.

Close modal

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

FIGURE 3.

Intracellular but not surface expression of FcεRIα 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 FcεRIα immunoprecipitated from eosinophil lysates with mAb 22E7 (lane 1); immunoprecipitation with an isotype-matched IgG control (lane 2); α-chain immunoprecipitated from Jurkat cells with mAb 22E7 (lane 3), used as negative controls. The membrane was immunoblotted with mAb 15A5 (FcεRI α-chain). B, The upper panel shows the presence of FcεRI α-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 FcεRIα on basophils, but not eosinophils after blotting with HRP-conjugated streptavidin. IP, immunoprecipitation; IB, immunoblotting.

FIGURE 3.

Intracellular but not surface expression of FcεRIα 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 FcεRIα immunoprecipitated from eosinophil lysates with mAb 22E7 (lane 1); immunoprecipitation with an isotype-matched IgG control (lane 2); α-chain immunoprecipitated from Jurkat cells with mAb 22E7 (lane 3), used as negative controls. The membrane was immunoblotted with mAb 15A5 (FcεRI α-chain). B, The upper panel shows the presence of FcεRI α-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 FcεRIα on basophils, but not eosinophils after blotting with HRP-conjugated streptavidin. IP, immunoprecipitation; IB, immunoblotting.

Close modal

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

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

FIGURE 4.

Western blot analysis of FcRγ (A and B) and FcεRIβ protein (C) in eosinophils. A, lane 1, Isotype-matched IgG was used as negative control for immunoprecipitation; lanes 2 and 3, clearly distinguishable FcRγ protein immunoprecipitated from eosinophil or basophil lysates with FcRγ-specific polyclonal rabbit antiserum (934); lane 4, FcRγ protein immunoprecipitated from Jurkat cells as negative controls. The membrane was immunoblotted with antiserum 934 to FcRγ. Data are representative of four separate experiments (donors 4, 5, 14, and 19) with similar results. B, Detectable FcRγ protein immunoprecipitated from surface-biotinylated eosinophils with FcRγ-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 FcεRIβ-specific polyclonal rabbit antiserum (976), showing the presence of FcεRI β-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.

FIGURE 4.

Western blot analysis of FcRγ (A and B) and FcεRIβ protein (C) in eosinophils. A, lane 1, Isotype-matched IgG was used as negative control for immunoprecipitation; lanes 2 and 3, clearly distinguishable FcRγ protein immunoprecipitated from eosinophil or basophil lysates with FcRγ-specific polyclonal rabbit antiserum (934); lane 4, FcRγ protein immunoprecipitated from Jurkat cells as negative controls. The membrane was immunoblotted with antiserum 934 to FcRγ. Data are representative of four separate experiments (donors 4, 5, 14, and 19) with similar results. B, Detectable FcRγ protein immunoprecipitated from surface-biotinylated eosinophils with FcRγ-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 FcεRIβ-specific polyclonal rabbit antiserum (976), showing the presence of FcεRI β-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.

Close modal

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

FIGURE 5.

Presence of FcεRIα 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 FcεRIα 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 FcεRI α-chain, as evidenced by FcεRIα immunoprecipitation from 1) eosinophil lysates after purification, 2) eosinophil lysates after 24 h in culture, and 3) eosinophil culture supernatants, with another FcεRI α-chain-specific mAb (15A5), followed by immunoblotting with the mAb 22E7 (donors 4 and 12). The relative amount of FcεRIα detected in eosinophils and their culture supernatant indicates that the soluble protein is not solely derived from eosinophil lysis.

FIGURE 5.

Presence of FcεRIα 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 FcεRIα 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 FcεRI α-chain, as evidenced by FcεRIα immunoprecipitation from 1) eosinophil lysates after purification, 2) eosinophil lysates after 24 h in culture, and 3) eosinophil culture supernatants, with another FcεRI α-chain-specific mAb (15A5), followed by immunoblotting with the mAb 22E7 (donors 4 and 12). The relative amount of FcεRIα detected in eosinophils and their culture supernatant indicates that the soluble protein is not solely derived from eosinophil lysis.

Close modal

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 FcεRI 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 FcεRI on peripheral blood eosinophils (16, 34). This discrepancy raised the question of whether the expression of FcεRI 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 FcεRIα, 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 FcεRIα 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-FcεRIα 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 α subunit in tissue-dwelling eosinophils (18, 34, 36, 37, 39, 40). In the study by Terada et al., no surface expression of FcεRIα was detected on peripheral blood eosinophils. However, double-labeling immunostaining of nasal biopsies demonstrated the presence of FcεRIα+ 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 α-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 α-chain mRNA and protein in late-phase BAL eosinophils (38). In their study, 85–95% of FcεRIα+ 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 FcεRIα 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 FcεRI 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 FcεR (unpublished observations).

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

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

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

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

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.

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.

3

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

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